Multifunctional cytokines

ABSTRACT

The present invention relates to a novel fusion protein with the formula X-Y, or Y-X, wherein X represents a first immunoregulating polypeptide and Y represents a second immunoregulating polypeptide different from X. The present invention also relates to a nucleic acid molecule encoding such a fusion protein and a vector comprising such a nucleic acid molecule. The present invention also provides infectious viral particles and host cells comprising such a nucleic acid molecule or such a vector as well as a process for producing such infectious viral particles. The present invention also relates to a method for recombinantly producing such a fusion protein. Finally, the present invention also provides a pharmaceutical composition comprising such a fusion protein, a nucleic acid molecule, a vector, infectious viral particles and a host cell as well as the therapeutic use thereof.

The present invention relates to a novel fusion protein with the formulaX-Y, or Y-X, wherein X represents a first immunoregulating polypeptideand Y represents a second immunoregulating polypeptide different from X.The present invention also relates to a nucleic acid molecule encodingsuch a fusion protein and a vector comprising such a nucleic acidmolecule. The present invention also provides infectious viral particlesand host cells comprising such a nucleic acid molecule or such a vectoras well as a process for producing such infectious viral particles. Thepresent invention also relates to a method for recombinantly producingsuch a fusion protein. Finally, the present invention also provides apharmaceutical composition comprising such a fusion protein, a nucleicacid molecule, a vector, infectious viral particles and a host cell aswell as the therapeutic use thereof.

The present invention is particularly useful in the field of genetherapy and immunotherapy, especially for treating or preventing avariety of diseases, including cancers and infectious diseases (bacteriaand virus infections).

Broadly speaking, host's immune responses fall into two categories:nonspecific (or innate) and specific (or adaptive or acquired). Thedifferences between these is that an specific immune response is highlyspecific for a particular antigen whereas nonspecific response does notrely on a repeated exposure to a given pathogen/antigen. The networkscontrolling the immune system rely on secreted proteins (e.g. cytokines)to turn on and off the functions of immune cells as well as to regulatetheir proliferation and to control the magnitude of the immune response.Specifically, two types of lymphocytes—B and T cells—are at the core ofspecific immunity. Upon being triggered by an antigen, B cells divideand the daughter cells synthesize and secrete antibody molecules(humoral immunity). T cell activation entails development ofcell-mediated immunity, mediated among others by cytotoxic T lymphocytes(CTL) that specifically eliminates non-self antigen-bearing target cells(e.g. infected or tumoral cells). Activation of a specific (oradaptative) immune response is orchestrated by numerous cytokines. Ofparticular importance are interleukin (IL)-1, IL-2, IL-6, IL-7, IL-15and interferon gamma (IFNg). On the other hand, nonspecific (innate)responses involve different types of immune cells, including naturalkiller (NK) cells, Natural Killer T cells (NKT), dendritic cells (DCs)and macrophages, and are among others mediated by the secretion ofcytokines such as IL-2, IL-12, IL-15, IL-18 and IL-21. In reality,however, a strict distinction between specific and nonspecific immuneresponses is somewhat arbitrary, as the elimination of pathogens andtumors in viva is likely to involve both types of immune responsesacting in concert. Also, through cytokine signalling pathways, specificeffectors may play a major role in the induction and activation ofnonspecific effectors and vice versa. For example, one striking propertyof NKT cells is their capacity to rapidly produce large amounts ofcytokines in response to T-cell receptor engagement, suggesting thatactivated NKT cells can also modulate specific immune responses. For ageneral discussion of immune response, immune effector cells and immunemediators, see for example the most updated editions of “Encyclopedia ofImmunology” (Edited by Ivan Roitt and Peter Delves; Academic PressLimited) and “Fundamental Immunology (e.g. 2^(nd) edition, Edited by W.Paul; Raven Press).

It is generally accepted that cancer is a multistep process whichresults from a loss of the control of cell multiplication. An extensivebody of research exists to support the involvement of tumor-associatedantigens (TAAs) in the onset of the malignant phenotype. These antigensinclude oncogene products (e.g. p53, ras, neu, erb), reactivatedembryonic gene products (e.g. P91A found in P815 mastocytoma), modifiedself-antigens (e.g. hyperglycosylated MUC-1), oncogenic viral genes(e.g. early antigens of papillomavirus) and a variety of others. Withregard to the mechanism that operates in the recognition and eliminationof tumor cells, it has been shown that T lymphocytes play a key role inconferring specificity to tumor rejection. In particular, CD8+ cytotoxicT lymphocytes (CTL) were identified as important effector cells forrecognizing specific tumor antigens. CTLs can kill tumors only afterthey have been presensitized to a tumor antigen and only when it ispresented at the cell surface by MHC class I gene products. In manycases, the induction of the anti-tumoral response is also dependent onthe presence of CD4+ T cells. In addition to these specific immuneeffector cells, roles have been identified in tumor rejection for NKcells and other nonspecific effector cells such as NKT and macrophages,which can lyse tumor cells in a manner that is not antigen-dependent andnot MHC-restricted.

Despite the fact that the vast majority of tumor-associated antigens iscapable of being recognized as foreign by the immune system of thepatient and the abundance of tumoricidal immune mechanisms, most cancersdo not provoke immunological responses sufficient to control the growthof malignant cells. Tumor cells have developed several mechanisms whichenable them to escape host immunity due to a reduction in antigenpresentation by the tumor cells or due to a generalized decline inpatient's immunity. As the expression of MHC class I determinants oncell surface is essential for the recognition of foreign antigens byCTLs, suppression or failure to express MHC class I antigens is one ofthe documented mechanisms used by tumor cells to evade the immune system(Tanaka et al., 1988, Ann. Rev. Immunol. 6, 359-380). Another mechanismof immune anergy involves the shedding of tumor antigens, thuspreventing the interaction of the immune cells with the tumor targetcell itself. Moreover, tumors can activate immunosuppressive moleculesto dampen the vigor of immune responses to tumor antigens or to activateapoptosis of immune effector cells. For example, IL-2 may have in somecircumstances, a critical role in the maintenance of peripheraltolerance. As a result of its pivotal role in activation-induced celldeath (AICD), the T cells generated in response to tumour vaccinescontaining IL-2 may interpret the tumor cells as self and thetumor-reactive T cells may be killed by AICD-induced apoptosis (Lenardo,1996, J. Exp. Med. 183, 721-724). Furthermore, IL-2 maintains CD4⁺CD25⁺negative regulatory T cells and has been reported to terminate CD8⁺memory T cell persistence (Shevach, 2000, Ann. Rev. Immunol. 18,423-449).

A number of studies have documented a critical role for tumor-specificCD4(+) cells in the augmentation of immunotherapeutic effectormechanisms. However, chronic stimulation of such CD4(+) T cells oftenleads to the up-regulation of both Fas and Fas ligand, and coexpressionof these molecules can potentially result in activation-induced celldeath (AICD) and the subsequent loss of anti-tumor response. Bycontrast, resistance to AICD significantly enhances T cell effectoractivity (Saff et al. 2004, J. Immunol. 172, 6598-6606).

A number of previous approaches have used cytokines to enhance host'simmunity, and thus to overcome tumor-induced state of immune anergy. Forexample, human IL-2 (Proleukin) is an approved therapeutic foradvanced-stage metastatic cancer. However, the systemic administrationof cytokines is often poorly tolerated by the patients and is frequentlyassociated with a number of side-effects including nausea, bone pain andfever (Mire-Sluis, 1993, TIBTech vol. 11; Moore, 1991, Ann Rev Immunol.9, 159-191). These problems are exacerbated by the dose levels that arerequired to maintain effective plasma concentrations. Cytokine deliveryusing virus vectors and cell vehicles have been proposed to reducesystemic toxicity.

Genetically modified tumor cells releasing various cytokines have beenshown to enhance tumor immunogenicity and to induce the regression ofpre-existing tumors. Immunization with tumor cells modified to secreteIL-2 (Karp et al., 1993, J. Immunol. 150, 896-908), alpha interferon(IFNa) (Porgador et al., 1993, J. Immunol. 150, 1458-1470) or GM-CSF(Dranoff et al., 1993, PNAS 90, 3539-3543) have been shown to enhancetumor immunogenicity and to induce the regression of preexisting tumors.In some instances, immunological memory has been generated to resist thesubsequent challenge with unmodified, parental tumor cells. Moreover,cytokine-transduced tumors may attract an inflammatory exudate in vivothat generally results in tumor destruction in animal models.Experimental animals and a small number of patients with establishedneoplasms treated with the cytokine-secreting tumor cells survived for alonger period of time, although in most instances tumor-growtheventually recurred.

The direct injection into solid tumors of vectors carrying genesencoding a variety of cytokines and chemokines has also been attemptedin order to enhance the presentation of T-cell epitopes or to enhancethe activation of tumor-specific T-lymphocytes. Many cytokines,including gamma interferon (IFN-g), IL-2 (Slos et al., 2001, Cancer GeneTher. 8, 321-332), IL-7 (Miller et al., 2000, Human Gene Therapy 11(1),53-65; Sharma et al., 1996, Cancer Gene Therapy 3, 302-313), IL-12(Melero et al., 2001, Trends Immunol. 22, 113-115), IL-15 (Suzuki etal., 2001, J. Leukoc. Biol. 69, 531-537; Kimura et al., 1999, Eur. J.Immunol. 29, 1532-1542), IL-18 (Cao et al., 1999, FASEB J. 13,2195-2202), and IL-21 (Ugai et al., 2003, Cancer Gene Therapy 10,187-192) have demonstrated significant antitumor activity in mice. Forexample, intra-tumoral injection of dendritic cells transduced with anadenovirus expressing IL-7 leads to significant systemic immuneresponses and potent anti-tumor effects in murine lung cancer models(Miller et al., 2000, Hum Gene Ther. 11, 53-65).

More recently, many studies with both mouse and human tumor models haveshown the importance of cytokine combinations in the development ofoptimal immune responses (see for example Putzer et al., 1997, Proc NatlAcad Sci USA. 94, 10889-10894; Melero et al., 2001, Trends Immunol. 22,113-115; Zhu et al., 2001, Cancer Res. 61, 3725-3734). For example, thecombination of IL-12 with the Th1 promoting IL-18 has been shown usefulfor the stimulation of the cell-mediated immune response (Hashimoto etal., 1999, J. Immunol. 163, 583-589; Barbulescu et al., 1998, J.Immunol. 160, 3642-3647). IL-2 and IFNg have been shown to cooperate forinhibiting tumor cell growth (U.S. Pat. No. 5,082,658). More recently,IL-21 was described to synergize the effects of IL-15 or IL-18 in theenhancement of 11-Ng production in human NK and T cells (Strengell etal., 2003, J. Immunol., 170, 5464-5469). The combination of IL-4 andGM-CSF is particularly useful in stimulating DCs (Palucka et al., 1998,J. Immunol. 160, 4587-4595). In other studies, it was found that thecombination of IL-3 and IL-11 had a synergistic effect with IL-12 on theproliferation of early hematopoktic progenitor cells (Trinchieri et al.,1994, Blood 84, 4008-4027). Graham and colleagues pioneered thecombination of two adenoviruses, one encoding IL-2 and the other IL-12(Addison et al., 1998, Gene Ther. 5, 1400-1409). They observed completeregression in more than 60% of established mammary carcinomas andinduction of potent antitumor CTL activity. Recent data show that IL-15can also synergize with IL-12 after double-transfection of human lungcancer cells (Di Carlo et al., 2000, J. Immunol. 165, 3111-3118). Also,IL-18 has been identified as a potent inducer of IFNg, and importantly,upregulates the expression of IL-12 receptors (Nakanishi et al., 2001,Ann. Rev. Immunol. 19, 423-474). In a reported poorly immunogenic tumor(MCA205), a clear synergy between these two cytokines was observed withantitumor effects mainly mediated by NK, cells.

However, in many of these studies, it was found that the relative levelof each cytokine was very important. For example, synergy studiesbetween IL-12 and other cytokines for the generation of antitumorresponses in mice have shown mixed results. Whereas the addition ofIL-12 in the presence of suboptimal amounts of IL-2 led to synergy inthe induction, proliferation, cytolytic activity and IFNg induction,combinations of IL-2 and IL-12 using a high dose of one cytokine werefound to be antagonistic (Perussia et al., 1992, J Immunol. 149,3495-3502; Mehrotra et al., 1993, J. Immunol. 151, 2444-2452). In somemodels, a non-optimal dose of one cytokine with respect to the other ledto an enhanced toxicity, while in other models, combinations of IL-12and IL-2 showed little or no synergy (e.g. Nastala et al., 1994, J.Immunol. 153, 1697-1706). A similar situation occurs with combinationsof IL-12 and IL-7. These results may reflect the inherent difficulty ofcombining two potentially synergistic cytokines in vivo, especially whenthere is a need to maintain a fixed ratio of activities of twocomponents with different pharmacological properties, such as differentcirculating half life and biodistribution.

To reduce the difficulties inherent to cytokine combinations, onestrategy is to fuse the cytokines. Fusions between two cytokines havealready been proposed in the literature. For example, WO 01/10912describes fusions between IL-12 and a second cytokine with short halflife in order to provide a longer pharmacokinetic behavior similar tothat of IL-12 itself. The fusion of IL-12 with either IL-2,granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-4 isspecifically disclosed. U.S. Pat. No. 5,883,320 and WO 92/04455 disclosefusions between IL-3 and a second cytokine, which may be used in thetreatment of diseases associated with a decreased level of hematopoïeticcells. The fusion between IL-3 and IL-11 was shown to be useful forstimulating the production of megakaryocytes and platelets. Drexler etal. (1998, Leuk Lymphoma 29, 119-128) describe the fusion of GM-CSF andIL-3. Finally, U.S. Pat. No. 6,261,550 envisages the fusion of G-CSFwith a cytokine to enhance hematopoïesis, e.g. to compensatehematopoïetic deficits resulting from chemotherapy or radiation therapyin cancer patients.

The development of efficient molecules against human tumors has been along sought goal which has yet to be achieved. In light of the forgoing,there remains a need for cytokine fusions which evoke an immune responseand are capable of bypassing tumor immunosuppression.

This technical problem is solved by the provision of the embodiments asdefined in the claims.

The present invention provides novel fusion proteins that are useful forenhancing an immune response, especially a specific together with anonspecific immune response in a host organism. The resulting responseis useful for reversing immunosuppression or anergy mechanisms inducedby pathogens or cancer cells. These fusion proteins or vectorsexpressing them can be used for protecting an animal or a human againsta variety of clinical conditions, such as acute or chronic infections orcancers. The present invention illustrates fusion proteins that providea high rate of tumor rejection after intratumoral delivery of adenoviralvectors encoding them into various animal models, providing evidence forsignificant immunostimulation. In accordance with the present invention,these fusion proteins or their encoding sequences may also be used asimmunoadjuvant to vaccine technologies (e.g. in combination with one ormore immunogen(s)) or in combination with suicide gene approaches, inthe prevention and treatment of cancer or infectious diseases in humansand other mammals.

Accordingly, in a first aspect, the present invention provides a novelfusion protein with the formula:

X-Y, or  a)

Y-X,  b)

wherein X represents a first immunoregulatory polypeptide;

Y represents a second immunoregulatory polypeptide; and

X is different from Y.

As used herein throughout the entire application, the terms “a” and “an”are used in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced compounds or steps,unless the context dictates otherwise. For example, the term “a cell”includes a plurality of cells including a mixture thereof.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

As used herein, when used to define products, compositions and methods,the teen “comprising” is intended to mean that the products,compositions and methods include the referenced components or steps, butnot excluding others. “Consisting essentially of” shall mean excludingother components or steps of any essential significance. Thus, acomposition consisting essentially of the recited components would notexclude trace contaminants and pharmaceutically acceptable carriers.“Consisting of” shall mean excluding more than trace elements of othercomponents or steps.

The term “polypeptide” or “protein” are used herein interchangeably torefer to polymers of amino acids of any length, preferably of at least50 amino acid residues. The polymer may be linear or branched, it maycomprise modified amino acids, and it may be interrupted by non-aminoacids. The term also encompasses an amino acid polymer that has beenmodified in one or more amino acid residue(s) by way of substitution oraddition of moieties or by chemical modification techniques well knownin the art. Included within the scope of the present invention are forexample disulfide bond formation, glycosylation, lipidation,hydroxylation, iodination, methylation, acetylation, acylation, gammacarboxylation, phosphorylation, proteolytic processing, or any othermanipulations such as conjugation or binding with a detectable moiety(i.e. a scintigraphic, radioactive, fluorescent, or dye labels and thelike). Suitable radioactive labels include but are not limited to^(99m)Tc, ¹²³I and ¹¹¹In. In the context of this invention, the terms“amino acid” and “residue” are synonyms. They encompass natural,unnatural and/or synthetic amino acids, including D or L opticalisomers, modified amino acids and amino acid analogs.

The term “fusion” or “fusion protein” or “fusion cytokine” as usedherein refers to the combination of amino acid sequences of the firstpolypeptide and of the second polypeptide in one polypeptide chain,preferably by in frame fusion of corresponding coding nucleotidesequences. In the nature, the X and Y entities may normally exist inseparate proteins, which are brought together in the fusion protein ofthe invention. In the fusion protein of the present invention, thecoding sequence of the first polypeptide (X) is fused in frame with thecoding sequence of the second polypeptide (Y) either directly or througha linker. By “fused in frame” is meant that the expression of the fusedcoding sequences results in the fusion protein comprising both the firstand the second polypeptides. This means for example that there is notranslational terminator between the reading frames of the X and Ypolypeptides. Even through the fusion between the X and Y entities cantake place internally at any site, the Y entity is preferably fused toeither the COOH or the NH2 terminus of the X entity (resulting in afusion of the formula X-Y and Y-X respectively). As used herein, theterm “directly” refers to a fusion of the polypeptides X and Y without apeptide linker in between (i.e. the codons encoding the X entity arecontiguous to the codons encoding the Y entity). In addition, the fusionprotein may also include further elements apart from X, Y and a linker,such as an initiator methionine, a signal peptide and/or a propeptide.Fusion proteins essentially consisting of or consisting of X and Y, andoptionally a linker, are preferred embodiments in the context of thepresent invention.

The term “immunoregulatory polypeptide” as used herein refers to apolypeptide capable of regulating an immune response in an animal orhuman organism. “Regulating an immune response” refers to modulating theactivity of immune effector cells or mediator molecules involved in animmune response. The term “regulate” can refer to enhancing or reducingan immune response, with a special preference for an enhancement. Asused herein the tens “enhancing” refers to inducing the onset and/ormodulating the magnitude and duration of an immune response leading tothe activation, differentiation, maturation and/or proliferation of oneor more immune effector cells and/or to the production of appropriateimmune mediators, and/or to the improvement of antigen presentation,and/or to the onset of a clinical benefit (e.g. inhibition of tumorgrowth, tumor regression). Regulation of an immune response can bedetermined using methods known in the art as well as methods disclosedherein.

The fusion protein of the invention “enhances” an immune response whenthe immune response—whether specific or nonspecific—observed with theaddition of the fusion protein is greater or intensified in any way whencompared to the same immune response measured without its addition.Preferably, the enhancement of the immune response provided by thefusion protein of the invention leads to the amelioration of a diseasecondition. The ability of the fusion polypeptide of the invention toenhance an immune response can be evaluated either in vitro or in vivousing a variety of assays which are standard in the art. For a generaldescription of techniques available to evaluate the onset and activationof an immune response, whether specific or non specific, see for exampleColigan et al. (1992 and 1994, Current Protocols in Immunology; ed JWiley & Sons Inc, National Institute of Health; incorporated herein byreference). Testing and validation of the fusion proteins of theinvention are also illustrated in the appended Example section. Suitableassays include without limitation the determination of the activationstatus for a particular type of immune effector cells, the proliferationrate of such cells, the quantification of the cell-surface markers, thelytic activity of the immune effector cells towards appropriate tumor ortarget cells, the measurement of cytokine expression profiles secretedby the activated effector cells. Suitable methods for proceeding to theevaluation of an immune response are conventional and include amongothers ELISA, immunofluorescence, Western blotting,immunohistochemistry, histology, flow cytometry (FACS). For example, Tcell proliferation can be determined, e.g. by a classical [³H]thymidineuptake assay. As another example, the lytic activity of cytotoxic Tcells can be measured, e.g. using a ⁵¹Cr release assay, with and withoutthe fusion protein. Naive and activated immune effector cells can alsobe discriminated by the identification of specific cell surface markers.For example, immature or nave T cells may be identified by theirexpression of the high molecular weight isoform of the CD45 moleculeknown as CD45RA. Mature T cells express the low molecular weight isoformof CD45 known as CD45RO. Upregulation of CD80, CD86 and MHCII-Iabreflects maturation of dendritic cells. The presence of CD8 is a markerof activated CTLs. Other informative markers of the type ormaturation/activation status of these immune cells are known in the art.Suitably, the candidate fusion protein can also be tested in anappropriate animal model to evaluate its anti-tumor activity, reflectingan enhancement of the immune response. For example, the fusion proteincan be administered into tumor animal models and the tumor growth and/orthe survival rate are evaluated periodically as compared to a control.In addition to in vivo methods for determining tumor inhibition, avariety of in vitro methods may be utilized in order to predict in vivotumor inhibition. Representative examples include lymphocyte mediatedanti-tumor cytolytic activity determined, for example, by a ⁵¹Cr releaseassay, tumor dependent lymphocyte proliferation (Ioannides et al., 1991,J. Immunol. 146, 1700-1707), in vitro generation of tumor-specificantibodies (Herlyn et al., 1984, J. Immunol. Meth. 73, 157-167), cell(e.g., CTL, helper T cell) or humoral (e.g., antibody)-mediatedinhibition of cell growth in vitro (Gazit et al., 1992, Cancer Immunol.Immunother. 35; 135-144) and determination of cell precursor frequency(Vose, 1982, hit. J. Cancer 30, 135-142).

In a preferred embodiment, the fusion protein of the invention providesan enhancement of the immune response as compared to the correspondingimmune response when said fusion protein is not added, by a factor of atleast 2, more preferably by a factor of at least 3.

The fusion proteins encompassed by the present invention are not limitedby the particular identity of X and Y, nor by the number of X and/or Yentities employed in the fusion protein. The X and the Y polypeptidesare different, i.e. heterologous with respect to one another. Thedifference may be in terms of structure (e.g. below 40% of identitybetween their respective amino acid sequence) and/or in terms of theirrespective biological activity (e.g. X and Y are involved in differentpathways of the immune system). The X and Y entities involved in thefusion protein of the invention may individually originate (be obtained,isolated) from human or animal origin (e.g. canine, avian, bovine,murine, ovine, porcine, feline, simian and the like). The fusion proteinmay also comprise X and Y entities of diverse origins (e.g. X of humanorigin and Y of animal origin).

In a preferred embodiment, X represents an immunoregulatory polypeptidecapable of enhancing a specific immune response, whereas Y represents animmunoregulatory polypeptide capable of enhancing a nonspecific immuneresponse.

According to a preferred embodiment, the X and Y immunoregulatorypolypeptides in the above formulae each represents a cytokine. As usedherein, “cytokine” refers to a polypeptide that generally acts as amediator of immunity being specific and/or non specific. It will beappreciated that the present invention aims at providing a“multifunctional” fusion cytokine capable of inducing or enhancing animmune response in a host cell or organism, thus allowing to reduce orinhibit at least one mechanism of immune anergy that has been developedby tumor or infected cells to escape host immunity.

In accordance with the general goal of the present invention, Xpreferably represents a cytokine capable of enhancing a nonspecific(innate) immune response, especially an immune response mediated by oneor more of the effector cells selected from the group consisting ofmacrophages, dendritic cells, NK cells and NKT cells. Y preferablyrepresents a cytokine capable of enhancing a specific (adaptative)immunity, especially an immune response mediated by effector cells suchas B and/or T lymphocytes (CD4+ and/or CD8+ T cells).

A non-exclusive list of cytokines which are comprised by the definitionof X and/or Y includes the interleukins (IL), interferons (IFN),chemokines, Tumor Necrosis Factor receptor ligands (e.g. 4-1BBL, OX40L,GITRL), KIR (Killing Inhibitory Receptor) ligands, KAR (Killingactivatory Receptor) ligands (e.g. RAE-1 as disclosed in Genbankaccession number AF346595) and H60; see for example Diefenbach et al.,2001, Nature 413, 165-171; Diefenbach et al., 2003, Eur. J. Immunol. 33,381-391), IRFs (IFN regulatory factors) (e.g. IRF-3 as disclosed inGenbank accession number NM001571), IRF-7 as disclosed in Genbankaccession number U53830 or chimeras thereof; see for example Au et al.,1995, Proc. Natl. Acad. Sci. USA 92, 11657-11661; Zhang and Pagano,1997, Mol. Cell. Biol. 17, 5748-5757; Nguyen, et al., 1997, CytokineGrowth Factor Rev. 8, 293-312; Duguay et al., 2002, Cancer Res. 62,5148-5152; Sharma et al., 2003, Science 300, 1148-1151; Bramson et al.,2003, Vaccine 21, 1363-1370) and B cell stimulatory factors. X and Y mayinclude independently, without limitation, precursor, mature forms,variants of cytokines. Appropriate cytokines include without limitationIL-1 through IL-31, and IFNs alpha through gamma. It will be appreciatedthat these cytokines and the methods available to quantify their levelsin a given medium are described in basic text books such as Oppenheim etal. (2001, Cytokine Reference; A compendium of cytokines and othermediators of host defense; Eds Durum et al. Academic Press). Preferredfusion proteins are those wherein X and Y are independently IL-2, IL-7,IL-15, IL-18, IL-21, IL-27, IL-31 or IFNg. Preferably Y is not GM-CSFwhen X is IL-2 and Y is not IL-2, GM-CSF or IL-4 when X is IL-12.

IL-2 is a pleiotropic cytokine acting both in specific and non specificimmunity. After more than 20 years of research, it has been establishedthat IL-2 is a potent growth and differentiation factor for T cells.IL-2 also stimulates the cytolytic activity of NK cells (Caligiuri etal., 1990, J. Exp. Med. 171, 1509-1526) and of the so-called lymphocyteactivated killer (LAK) cells (Pawelec et al., 1999, Crit. Rev Oncog. 10,83-127). IL-2 induces the secretion of other cytokines including IFN-g(Trinchieri et al., 1984, J. Exp. Med. 160, 1147-1169). IL-2 also showsstrong B cell growth factor activity and can stimulate monocyte-lineagecells. IL-2 appears to be produced exclusively by antigen-activated Tlymphocytes including both CD4+ and CD8+ T cells. IL-2 mediates itsbiological activities by binding to IL-2 receptors (IL-2R), which areexpressed transiently on antigen-activated T cells and continuously byNK cells. The mature human IL-2 protein consists of 133 amino acids(Taniguchi et al., 1983, Nature 302, 305-310). It is synthesized as aprecursor containing 153 amino acids with a 20-residue hydrophobicleader sequence (signal peptide) that is cleaved to produce the matureprotein prior to or during secretion. The amino acid and nucleotidesequence of IL-2 from 31 species are now well known. For example, thesequence of human IL-2 protein in NCBI Genbank under accession numberP01585. Genbank accession numbers NM008366 and NM000586 describe themouse and human IL-2 gene sequences, respectively (all accession numbersincorporated herein by reference).

IL-7 plays an essential role in the development of T and B cells. Italso plays a role in differentiation of these cells. IL-7 stimulates thegrowth of immature and mature T cells, affects survival andproliferation of mature T cells, and promotes the expansion and effectorfunctions of cytolytic T cells and their precursors. Additionally, IL-7enhances LAK cell activity in peripheral blood and can stimulate theanti-tumor activity of monocytes and macrophages. IL-7 alsodown-regulates both macrophage and tumor production of TGFβ and thus mayserve to limit tumor-induced immune anergy (Dubinett et al., 1993, J.Immunol. 151, 6670-6680; Miller et al., 1993, Blood 82, 3686-3694). IL-7is a single chain glycosylated protein produced predominantly byepithelial cells, especially keratinocytes and thymic epithelial cells.The human IL-7 cDNA contains an open reading frame encoding a protein of177 amino acids including a 25 amino acid signal peptide which iscleaved from the mature protein during the secretion process. The DNAand amino acid sequences of IL-7 from a number of species are now wellknown (see for example Namen et al., 1988, J. Exp. Med. 167, 988-1002;Namen et al., 1988, Nature, 333, 571-573; Conlon et al., 1989, Blood 74,1368-1373). For example, the sequences of human, bovine and murine IL-7proteins are disclosed in GenEMBL under accession numbers NP000871,CAA45838 and CAA30779, respectively. The nucleotide sequence of themouse IL-7 gene is available in Genbank under accession number NM008371.The nucleotide sequence of the human IL-7 gene is available underaccession number NM000880. The bovine IL-7 gene is disclosed underaccession number X64540 (all accession numbers incorporated herein byreference). It will be appreciated that human (152 amino acids) andmurine (127 amino acids) IL-7 show 60% sequence homology at the proteinlevel.

Like IL-2, IL-15 is a pleiotropic cytokine acting both in specific andnonspecific immunity. The human IL-15 cDNA encodes a 162 amino acidprecursor consisting of a 48 amino acid leader peptide and a 114 matureprotein (Grabstein et al., 1994, Science 264, 965-968). IL-15 exerts itsbiological activities through binding to the IL-2R beta and gammachains, supplemented by a specific IL-15R alpha chain (Giri et al.,1995, EMBO J. 14, 3654-3663). This sharing of receptor subunits probablyaccounts for the similar functional activities of IL-2 and IL-15observed on T, B and NK, cells. IL-15 like IL-2 has been defined as a Tcell growth factor (Grabstein et al., 1994, Science 264, 965-968;Nishimura et al., 1996, J. Immunol. 156, 663-669). One of the mostcritical functions of IL-15 is its pivotal role in the development,survival and activation of NK cells. Treatment of NK cells with IL-15results in the proliferation and enhancement of cytotoxic activity andin the production of IFN-g, TNFa and GM-CSF (Carson et al., 1994, J.Exp. Med. 180, 1395-1403). Apart from its activities on T and NK cells,IL-15 costimulates, in a comparable way as IL-2, the proliferation ofactivated B cells (Armitage et al., 1995, J. 1 mmol. 154, 483-490).IL-15 promotes the generation and persistence of CD4+ memory cells (WO98/36768). The most striking differences, however, between IL-15 andIL-2 reside in their expression pattern. Contrary to IL-2, IL-15 mRNA iswidely distributed in a variety of non-lymphoid tissues such asfibroblasts and epithelial cells. On the other hand, it is not presentin resting or activated T cells, the predominant source of IL-2.Grabstein et al. (1994, Science 264, 965-968) provides disclosurerelating to obtaining the sequence for human IL-15. Genbank accessionnumbers NM008357 and NM000585 provide the mouse and human. IL-15nucleotide sequences, respectively. Accession numbers in GenEMBL forIL-15 amino acid sequences are: human protein (P40933), murine protein(P48346), rat protein (P97604) and bovine protein (Q28028) (allaccession numbers incorporated herein by reference).

IL-18 is a recently discovered Th1 cytokine that was described as havingsignificant immunoregulatory fractions on both T and NK cells (Okamuraet al., 1995, Nature 378, 88-91). In particular, IL-18 augments theproliferation of T cells, enhances cytotoxic activity of NK cells,induces secretion of GM-CSF from both NK and T cells, and synergizeswith IL-12 in terms of IFN-g production (Okamura et al., 1998, Curr OpinImmunol. 10, 259-264). IL-18 is synthesized as a biologically inactiveprecursor molecule (pro-IL-18). To generate the active form of IL-18,the pro sequence needs to be cleaved by the intracellular cysteineprotease, IL-1beta converting enzyme ICE, at the Asp-X processing site.IL-18 can inhibit tumor growth in some murine tumor systems, butregression of established tumor by IL-18 gene therapy alone has not beendemonstrated (Micallef et al., 1997, Cancer Immunol Immunother. 43,361-367; Osaki et al., 1998, J. Immunol. 160, 1742-1749; Osaki et al.,1999, Gene Ther. 6, 808-815; Hashimoto et al., 1999, J. Immunol. 163,583-589). The DNA and protein sequences of the IL-18 molecule arepublished (see for example Okamura et al., 1995, Nature 378, 88-91;Ushio et al., 1996, J. Immunol., 156, 4274-4279; Genbank accessionnumbers NM008360 and NM001562 describing respectively the mouse andhuman IL-18 nucleotide sequences, and NP_(—)001553 for the human IL-18protein; all accession numbers incorporated herein by reference).

IL-21 is a recently identified cytokine with a four-helix-bundlestructure (Parrish-Novak et al., 2000, Nature 408, 57-63). Theexpression and function of this cytokine and its receptor suggest thatIL-21 is a new player in lymphoid differentiation. IL-21 was found tohave potent effects on all classes of lymphocytes: B, T and NK cells.One of the most interesting biological activities of IL-21 is tosubstantially increase the cytotoxic activity of mature NK cells,independently of proliferation. The DNA and protein sequences of theIL-21 molecule are disclosed in the literature (see for exampleParrish-Novak et al., 2000, Nature 408, 57-63; Genbank accession numbersNM021782 and NM021803 describing respectively the mouse and human IL-21nucleotide sequences, and NP_(—)065386 for the human IL-21 protein; allaccession numbers incorporated herein by reference).

One cytokine that is well recognized to play a central role incoordinating tumor immune responses is IFNg. IFNg is mainly produced byactivated lymphocytes and exerts its activities in specific immuneresponses. In this regard, IFN-g augments expression of the MHC class Imolecules in professional as well as non-professional antigen-presentingcells. It is involved in T and B lymphocyte proliferation anddifferentiation. Production of IFNg by T helper cells is a hallmark ofthe Th1-type phenotype. Thus, high-level production of IFN-g istypically associated with an effective host defense againstintracellular pathogens. The importance of IFNg in anti-tumor therapy isbased on its anti-angiogenic properties, and its ability todown-regulate the expression of immunosuppressive molecules secreted bytumors. By increasing tumor immunogenicity, IFNg ultimately enhancestumor recognition by tumor-specific cytotoxic T lymphocytes, and favorstumor rejection (Beatty et al., 2001, Immunol Res. 24, 201-10). The DNAand protein sequences of the IFNg molecule are disclosed in theliterature (see for example Gray et al., 1982, Nature 295, 503-508; Grayet al., 1983, Proc. Natl. Acad. Sci. USA 80, 5842-5846; Genbankaccession number K00083 describing the mouse IFNg gene sequence andGenbank accession number NM000619 describing the human IFNg genesequence, and II01579 for the human IFNg protein; all accession numbersincorporated herein by reference).

In a preferred aspect of the present invention, the fusion protein ofthe invention is a fusion protein wherein:

-   -   (a) X is IL-2 and Y is selected from the group consisting of        IL-7, IL-15, IL-18, IL-21, IL-27, IL-31 and IFN-g,    -   (b) X is IL-12 and Y is selected from the group consisting of        IL-15, IL-18 and IL-21,    -   (c) X is IL-15 and Y is selected from the group consisting of        IL-7, IL-18 and IL; 21; and    -   (d) X is IL-18 and Y is IL-21;

In the context of the present invention, the X and Y entities used inthe fusion proteins of the invention can be obtained (isolated orderived) from any species. Particularly preferred are fusions involvingeither the native or a biologically active modified form of the humancytokines. When referring to IL-12, it is mentioned that IL-12 can be inthe form of a heterodimeric protein composed of 35 and 40 kDa subunits(in this case the Y entity is fused either to the 35 or the 40 kDasubunit) or in the form of a monomeric protein where 35 and 40 kDasubunits are fused together as a single chain protein (in this case theY entity is fused to the 35-40 kDa fusion), this latter being preferredin the context of the present invention. Preferably, the IL-12 entity(p35 or p40 or p35-p40 single chain) is placed at the N-terminus of thefusion protein of the invention (e.g. IL-12/IL-15, IL-12/IL-21).

The conformation of the fusion may be important to reach the optimalactivity of the fusion protein of the invention. Accordingly, thepresent invention provides fusion proteins which comprise, oralternatively consist essentially of, or alternatively consist of afusion protein, which:

-   -   (a) has the formula Y-X, wherein X is IL-2 and Y is IL-7 (i.e.        wherein IL-7 is fused to the NH2-terminus of IL-2, said fusion        protein being designated IL-7/IL-2);    -   (b) has the formula X-Y, wherein X is IL-2 and Y is IL-15 (i.e.        wherein IL-15 is fused to the COOH-terminus of IL-2, said fusion        protein being designated IL-2/IL-15), or has the formula Y-X,        wherein X is IL-2 and Y is IL-15 (i.e. wherein IL-15 is fused to        the NH2-terminus of IL-2, said fusion protein being designated        IL-15/IL-2);    -   (c) has the formula X-Y, wherein X is IL-2 and Y is IL-18 (i.e.        wherein IL-18 is fused to the COOH-terminus of IL-2, said fusion        protein being designated IL-2/IL-18);    -   (d) has the formula Y-X, wherein X is IL-2 and Y is IL-21 (i.e.        wherein IL-21 is fused to the NH2-terminus of IL-2, said fusion        protein being designated IL-21/IL-2);    -   (e) has the formula Y-X, wherein X is IL-2 and Y is IFNg (i.e.        wherein IFNg is fused to the NH2-terminus of IL-2, said fusion        protein being designated IFNg/IL-2);    -   (f) has the formula X-Y, wherein X is IL-15 and Y is IL-7 (i.e.        wherein IL-15 is fused to the NH2 terminus of IL-7, said fusion        protein being designed IL-15/IL-7);    -   (g) has the formula X-Y, wherein X is IL-15 and Y is IL-18 (i.e.        wherein IL-18 is fused to the COOH-terminus of IL-15, said        fusion protein being designated IL-15/IL-18), or has the formula        Y-X, wherein X is IL-15 and Y is IL-18 (i.e. wherein IL-18 is        fused to the NH2-terminus of IL-15, said fusion protein being        designated IL-18/IL-15);    -   (h) has the formula X-Y, wherein X is IL-15 and Y is IL-21 (i.e.        wherein IL-21 is fused to the COOH-terminus of IL-15, said        fusion protein being designated IL-15/IL-21), or has the formula        Y-X, wherein X is IL-15 and Y is IL-21 (i.e. wherein IL-21 is        fused to the NH2 terminus of IL-15, said fusion protein being        designated IL-21/IL-15); or    -   (i) has the formula X-Y, wherein X is IL-18 and Y is IL-21 (i.e.        wherein IL-21 is fused to the COOH-terminus of IL-18, said        fusion protein being designated IL-18/IL-21) or has the formula        Y-X, wherein X is IL-18 and Y is IL-21 (i.e. wherein IL-21 is        fused to the NH2-terminus of IL-18, said fusion protein being        designated IL-21/IL-18).

As mentioned before, the present invention encompasses fusion proteinsinvolving full-length pre-processed forms, as well as mature processedforms, fragments thereof and variants of each or both of the X and Yentities, including allelic as well as non-naturally occurring variants.In addition to naturally-occurring allelic variants of the X and/or Yentities that may exist in the population, the skilled artisan willfurther appreciate that changes (i.e. one or more deletions, additionsand/or substitutions of one or more amino acid) can be introduced bymutation using classic or recombinant techniques to effect random ortargeted mutagenesis. A suitable variant in use in the present inventionpreferably has an amino acid sequence having a high degree of homologywith the amino acid sequence of the corresponding native cytokine. Inone embodiment, the amino acid sequence of the variant cytokine in usein the fusion protein of the invention is at least 70%, at least about75%, at least about 80%, at least about 90%, preferably at least about95%, more preferably at least about 97% and even more preferably atleast about 99% identical to the corresponding native sequence.

Percent identities between amino acid or nucleic acid sequences can bedetermined using standard methods known to those of skill in the art.For instance for determining the percentage of homology between twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes. The amino acid residues at corresponding amino acid positionsare then compared. Gaps can be introduced in one or both amino acidsequence(s) for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes. When a position in the firstsequence is occupied by the same amino acid residue as the correspondingposition in the second sequence, then the sequences are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps which need to be introduced foroptimal alignment and the length of each gap. The comparison ofsequences and determination of percent identity and similarity betweentwo sequences can be accomplished using a mathematical algorithm (e.g.Computational Molecular Biology, 1988, Ed Lesk A M, Oxford UniversityPress, New York; Biocomputing: Informatics and Genome Projects, 1993, EdSmith D. W., Academic Press, New York; Computer Analysis of SequenceData, 1994, Eds Griffin A. M. and Griffin H. G., Human Press, NewJersey; Sequence Analysis Primer, 1991, Eds Griskov M. and Devereux J.,Stockton Press, New York). Moreover, various computer programs areavailable to determine percentage identities between amino acidsequences and between nucleic acid sequences, such as GCG™ program(available from Genetics Computer Group, Madison, Wis.), DNAsis™ program(available from Hitachi Software, San Bruno, Calif.) or the MacVector™program (available from the Eastman Kodak Company, New Haven, Conn.).

Suitable variants of X and/or Y entities for use in the presentinvention are biologically active and retain at least one of theactivities described herein in connection with the corresponding nativecytokine. Preferably, the therapeutic effect (e.g. anti-tumor activity,by-pass of tumor-induced immune anergy) is preserved, although a givenfunction of the native cytokine(s) may be positively or negativelyaffected to some degree, e.g. with variants exhibiting reducedcytotoxicity or enhanced biological activity. Amino acids that areessential for a given function can be identified by methods known in theart, such as by site-directed mutagenesis. Amino acids that are criticalfor binding a partner/substrate (e.g. a receptor) can also be determinedby structural analysis such as crystallization, nuclear magneticresonance and/or photoaffinity labeling. The resulting variant can betested for biological activity in assays such as those described above.

For example, in one class of functional variants, one or more amino acidresidues are conservatively substituted. A “conservative amino acidsubstitution” is one in which the amino acid residue in the nativepolypeptide is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art (see for example the matrix of FIGS. 84 and 85of the Atlas of Protein Sequence and Structure, 1978, Vol. 5, ed. M. O.Dayhoff, National Biomedical Research Foundation, Washington, D.C.).Typically, substitutions are regarded as conservative when thereplacement, one for another, is among the aliphatic amino acids Ala,Val, Leu, and Ile; the hydroxyl residues Ser and Thr; the acidicresidues Asp and Glu; the amide residues Asn and Gln; the basic residuesLys and Arg; or the aromatic residues Phe and Tyr. Alternatively, inanother embodiment, mutations can be introduced randomly along all orpart of a cytokine coding sequence, such as by saturation mutagenesis,and the resultant mutant can be screened for its biological activity asdescribed herein to identify mutants that retain at least therapeuticactivity.

In accordance with the present invention, particularly important areIL-2 variants which exhibit a reduced cytotoxicity as compared to thecorresponding native IL-2. Suitable IL-2 variants include withoutlimitation those described in European patent EP 673 257 and U.S. Pat.No. 5,229,109 (incorporated by reference herein) having an amino acidsubstitution within the B alpha helix formed by residues 33-46 of thehuman IL-2. Specific examples of low toxic IL-2 variants include thevariant F42K having the phenyl alanine residue in position 42 of thenative IL-2 substituted by a lysine residue, or the variant R38A havingthe arginine residue in position 38 of the native IL-2 substituted by analanine residue. Further IL-2 variants suitable for use in the presentinvention also include those described in WO 99/60128 and by Shanafeltet al. (2000, Nat Biotech 18, 1197-1202) (incorporated by referenceherein). Specific examples include the variant D20I having the asparticacid in position 20 of the native IL-2 substituted by an isoleucineresidue, the variant N88G having the asparagine in position 88 of thenative IL-2 substituted by a glycine residue, the variant N88R havingthe asparagine in position 88 of the native IL-2 substituted by anarginine residue and the variant Q126M having the glutamine in position126 of the native IL-2 substituted by a methionine residue or anycombination thereof. The term “in position” as used herein encompassesthe meaning that the respective cytokine variant is mutated at a sitecorresponding that of the position in the respectively cited nativecytokine.

Suitable IL-15 variants for use in the present invention include withoutlimitation those described in WO 02/63044 relating to genetic variantsof human IL-15 gene as well as any variant of IL-15 which is mutated atone or more amino acid residue(s) which interferes with the binding toalpha chain of IL-15 receptor (e.g. those described by Bernard et al.(2004, J. Biol. Chem. 279, 24313-22 with a special preference forvariants L45E, S51D, L52D, E64K, N65K and 168D, where the mutationpositions are indicated by reference to Bernard et al. with +1corresponding to the first residue of the mature IL-15).

In the context of the present invention, particularly important areIL-18 variants which exhibit an enhanced biological activity as comparedto the corresponding native IL-18. Suitable IL-18 variants includewithout limitation those described by Kim et al. (2002, J. Biol. Chem.277, 10998-11003) and Kim et al. (2001, Proc. Natl. Acad. Sci. USA 98,3304-3309) (incorporated by reference herein), and more particularly thevariant E42A having the glutamic acid residue in position 42 of thenative IL-18 substituted by an alanine residue or the variant K89Ahaving the lysine residue in position 89 of the native IL-18 substitutedby an alanine residue or a variant combining both substitutions.Preferably, the IL-18 comprising fusion proteins of the presentinvention involve a mutated IL-18 having the lysine in position 89 ofthe native IL-18 substituted by an alanine residue (K89A).

Moreover, as mentioned above, the term IL-18 as used herein encompassesboth proIL-18 and mature IL-18. According to one and preferredembodiment, the IL-18 polypeptide used in the present invention is apro-IL-18 (i.e. comprising its endogenous prosequence), especially whenit is fused to the NH2 terminus of the other cytokine partner. But, theuse of an IL-18 entity (e.g. mature IL-18) comprising an heterologous(with respect to IL-18) prosequence can also be envisaged. According toanother embodiment, the IL-18 polypeptide used in the present inventionlacks its prosequence, especially when it is fused to the COOH terminusof the other cytokine partner.

Although the X and Y entities can be directly fused in the fusionprotein of the invention, it is however preferred to use a linkerpeptide for joining X and Y. The purpose of the linker is to allow thecorrect formation, folding and/or functioning of each of the X and Yentities. It should be sufficiently flexible and sufficiently long toachieve that purpose. Preferably, the coding sequence of the linker maybe chosen such that it encourages translational pausing and thereforeindependent folding of the X and Y entities. A person skilled in the artwill be able to design suitable linkers in accordance with theinvention. The present invention is, however, not limited by the form,size or number of linker sequences employed. Multiple copies of thelinker sequence of choice may be inserted between X and Y. The onlyrequirement for the linker sequence is that it functionally does notadversely interfere with the folding and/or functioning of theindividual entities of the fusion protein. For example, a suitablelinker is 5 to 50 amino acid long and may comprise amino acids such asglycine, serine, threonine, asparagine, alanine and proline (see forexample Wiederrecht et al., 1988, Cell 54, 841; Dekker et al., 1993,Nature 362, 852; Sturm et al., 1988, Genes and Dev. 2, 1582; Aumailly etal., 1990 FEBS Lett. 262, 82). Repeats comprising serine and glycineresidues are preferred in the context of the invention. A specificexample of suitable linkers consists of two or three or more (e.g. up toeight) copies of the sequence Gly-Gly-Gly-Gly-Ser (GGGGS). It will beevident that the invention is not limited to the use of this particularlinker.

The invention further includes fusion proteins which comprise, oralternatively consist essentially of, or alternatively consist of anamino acid sequence which is at least 70%, 75%, 80%, 90%, 95%, 97%, 99%homologous or even better 100% homologous (identical) to all or part ofany of the amino acid sequences recited in SEQ ID NO: 1-19.

The sequence recited in SEQ ID NO:1 corresponds to the fusion betweenhuman IL7 and human IL-2, with the human IL-7 extending from amino acidresidue 1 to amino acid residue 177, the linker peptide extending fromamino acid residue 178 to amino acid residue 192, and the human IL-2extending from amino acid residue 193 to amino acid residue 345. Thesequence recited in SEQ ID NO: 2 corresponds to the fusion betweenmurine IL7 and murine IL-2, with the murine IL-7 extending from aminoacid residue 1 to amino acid residue 154, the linker peptide extendingfrom amino acid residue 155 to amino acid residue 164, and the murineIL-2 extending from amino acid residue 165 to amino acid residue 333.

The sequence recited in SEQ ID NO:3 corresponds to the fusion betweenhuman IL-2 and human IL-15, with the human IL-2 extending from aminoacid residue 1 to amino acid residue 153, the linker peptide extendingfrom amino acid residue 154 to amino acid residue 168, and the humanIL-15 extending from amino acid residue 169 to amino acid residue 330.The sequence recited in SEQ ID NO:4 corresponds to the fusion betweenhuman IL-15 and human IL-2, with the human IL-15 extending from aminoacid residue 1 to amino acid residue 162, the linker peptide extendingfrom amino acid residue 163 to amino acid residue 177, and the humanIL-2 extending from amino acid residue 178 to amino acid residue 330.The sequence recited in SEQ ID NO:5 corresponds to the fusion betweenthe signal peptide of human IL-2, human IL-15 and human IL-2, with thesignal peptide of human IL-2 extending from amino acid residue 1 toamino acid residue 20, the human IL-15 extending from amino acid residue21 to amino acid residue 182, the linker peptide extending from aminoacid residue 183 to amino acid residue 197, and the human IL-2 extendingfrom amino acid residue 198 to amino acid residue 350. The sequencerecited in SEQ ED NO:6 corresponds to the fusion between murine IL-2 andmurine IL-15, with the murine IL-2 extending from amino acid residue 1to amino acid residue 169, the linker peptide extending from amino acidresidue 170 to amino acid residue 179, and the murine IL-15 extendingfrom amino acid residue 180 to amino acid residue 324. The sequencerecited in SEQ ID NO: 7 corresponds to the fusion between murine IL-15and murine IL-2, with the murine IL-15 extending from amino acid residue1 to amino acid residue 145, the linker peptide extending from aminoacid residue 146 to amino acid residue 155, and the murine IL-2extending from amino acid residue 156 to amino acid residue 324.

The sequence recited in SEQ ID NO: 8 corresponds to the fusion betweenhuman IL-2 and human IL-18 (pro-IL-18), with the human IL-2 extendingfrom amino acid residue 1 to amino acid residue 153, the linker peptideextending from amino acid residue 154 to amino acid residue 168, and thehuman pro-IL-18 extending from amino acid residue 169 to amino acidresidue 361. The sequence recited in SEQ ID NO: 9 corresponds to thefusion between human IL-2 and the variant K89A of human pro-IL-18, withthe human IL-2 extending from amino acid residue 1 to amino acid residue153, the linker peptide extending from amino acid residue 154 to aminoacid residue 168, and the variant of human pro-IL-18 extending fromamino acid residue 169 to amino acid residue 361 with the amino acidresidue 257 being an alanine instead of a lysine in the native IL-18.The sequence recited in SEQ ID NO: 10 corresponds to the fusion betweenhuman IL-2 and human mature IL-18, with the human IL-2 extending fromamino acid residue 1 to amino acid residue 153, the linker peptideextending from amino acid residue 154 to amino acid residue 168, and thehuman mature IL-18 extending from amino acid residue 169 to amino acidresidue 325. The sequence recited in SEQ ID NO: 11 corresponds to thefusion between human IL-2 and the variant K89A of human mature IL-18,with the human IL-2 extending from amino acid residue 1 to amino acidresidue 153, the linker peptide extending from amino acid residue 154 toamino acid residue 168, and the variant of human mature IL-18 extendingfrom amino acid residue 169 to amino acid residue 325 with the aminoacid residue 221 being an alanine instead of a lysine in the nativeIL-18. The sequence recited in SEQ ID NO: 12 corresponds to the fusionbetween murine IL-2 and murine pro-IL-18, with the murine IL-2 extendingfrom amino acid residue 1 to amino acid residue 169, the linker peptideextending from amino acid residue 170 to amino acid residue 179, and themurine pro-IL-18 extending from amino acid residue 180 to amino acidresidue 371. The sequence recited in SEQ ID NO: 13 corresponds to thefusion between murine IL-2 and the variant K89A of the murine IL-18,with the murine IL-2 extending from amino acid residue 1 to amino acidresidue 169, the linker peptide extending from amino acid residue 170 toamino acid residue 179, and the variant of the murine IL-18 extendingfrom amino acid residue 180 to amino acid residue 371 with the aminoacid residue 266 being an alanine instead of a lysine in the nativeIL-18. The sequence recited in SEQ ID NO: 14 corresponds to the fusionbetween murine IL-2 and murine mature IL-18, with the murine IL-2extending from amino acid residue 1 to amino acid residue 169, thelinker peptide extending from amino acid residue 170 to amino acidresidue 179 and the murine mature IL-18 extending from amino acidresidue 180 to amino acid residue 336. The sequence recited in SEQ IDNO: 15 corresponds to the fusion between murine IL-2 and the variantK89A of the murine mature IL-18, with the murine IL-2 extending fromamino acid residue 1 to amino acid residue 169, the linker peptideextending from amino acid residue 170 to amino acid residue 179 and thevariant of the murine mature IL-18 extending from amino acid residue 180to amino acid residue 336, with the amino acid residue 231 being analanine instead of a lysine in the native IL-18.

The sequence recited in SEQ ID NO: 16 corresponds to the fusion betweenhuman IL-21 and human IL-2, with the human IL-21 extending from aminoacid residue 1 to amino acid residue 179, the linker peptide extendingfrom amino acid residue 180 to amino acid residue 194 and the human IL-2extending from amino acid residue 195 to amino acid residue 347. Thesequence recited in SEQ ID NO: 17 corresponds to the fusion betweenmurine IL-21 and murine IL-2, with the murine IL-21 extending from aminoacid residue 1 to amino acid residue 146, the linker peptide extendingfrom amino acid residue 147 to amino acid residue 156 and the murineIL-2 extending from amino acid residue 157 to amino acid residue 325.

The sequence recited in SEQ ID NO: 18 corresponds to the fusion betweenhuman IFNg and human IL-2, with the human IFNg extending from amino acidresidue 1 to amino acid residue 166, the linker peptide extending fromamino acid residue 167 to amino acid residue 181 and the human IL-2extending from amino acid residue 182 to amino acid residue 334. Thesequence recited in SEQ ID NO: 19 corresponds to the fusion betweenmurine IFNg and murine IL-2, with the murine IFNg extending from aminoacid residue 1 to amino acid residue 155, the linker peptide extendingfrom amino acid residue 156 to amino acid residue 165 and the murineIL-2 extending from amino acid residue 166 to amino acid residue 334.

In the context of the present invention, a protein “consists of” anamino acid sequence when the protein does not contain any amino acidsbut the recited amino acid sequence. A protein “consists essentially of”an amino acid sequence when such an amino acid sequence is presenttogether with only a few additional amino acid residues, typically fromabout 1 to about 50 or so additional residues. A protein “comprises” anamino acid sequence when the amino acid sequence is at least part of thefinal (i.e. mature) amino acid sequence of the protein. Such a proteincan have a few up to several hundred additional amino acids residues.Such additional amino acid residues can be naturally associated witheach or both entities contained in the fusion or heterologous aminoacid/peptide sequences (heterologous with respect to the respectiveentities). Such additional amino acid residues may play a role inprocessing of the fusion protein from a precursor to a mature form, mayfacilitate protein trafficking, prolong or shorten protein half-life orfacilitate manipulation of the fusion protein for assay or production,among other things. Preferably, the fusion proteins of the inventioncomprise a signal peptide at the NH2-terminus in order to promotesecretion in the host cell or organism. For example, the endogenoussignal peptide (i.e. naturally present in the cytokine present at theNH2 terminus of said fusion) can be used or alternatively a suitableheterologous (with respect to the cytokine in question) signal peptidesequence can be added to the cytokine entity present at the NH2 terminusof the fusion or inserted in replacement of the endogenous one.Suitably, when IL-15 is present at the NH2 terminus of the fusionprotein of the invention, a heterologous peptide signal (heterologouswith respect to IL-15) can be added to or can replace the native signalof IL-15, in order to promote or increase secretion in a given host.Suitable heterologous signal peptides include without limitation thesignal peptides of IL-2 and signal peptide of immunoglobulins (Ig) suchas the signal peptide of the Kappa light chain of a mouse IgG (Meazza etal., 2000, Int. J. Cancer 87, 574; Susukiet al., 2001, J. Leukoc. Biol.69, 531). An illustrative example of this embodiment is provided by thefusion protein recited in SEQ ID NO: 5. Alternatively, it is alsopossible to use the endogenous IL-15 peptide signal either the short orthe long form thereof (Kuryus et al., 2000, J. Biol. Chem. 275, 30653).In addition, the fusion protein may also be fused to a tag peptide, forexample, a peptide that facilitates identification and/or purification.

In the context of the invention, the fusion proteins of the inventioncan comprise cytokine entities of any origin, i.e. any human or animalsource (including canine, avian, bovine, murine, ovine, feline, porcine,etc). Although “chimeric” fusion proteins are also encompassed by theinvention (e.g. one cytokine entity of human origin and the other of ananimal source), it is preferred that each entity be of the same origin(e.g. both from humans).

The fusion proteins of the present invention can be produced by standardtechniques. Polypeptide and DNA sequences for each of the cytokinesinvolved in the fusion protein of the present invention are published inthe art, as are methods for obtaining expression thereof throughrecombinant or chemical synthetic techniques. In another embodiment, afusion-encoding DNA sequence can be synthesized by conventionaltechniques including automated DNA synthesizers. Then, the DNA sequenceencoding the fusion protein may be constructed in a vector and operablylinked to a regulatory region capable of controlling expression of thefusion protein in a host cell or organism. Techniques for cloning DNAsequences for instance in viral vectors or plasmids are known to thoseof skill in the art (Sambrook et al, 2001, “Molecular Cloning. ALaboratory Manual”, Laboratory Press, Cold Spring Harbor N.Y.). Thefusion protein of the invention can be purified from cells that havebeen transformed to express it as described below.

The fusion protein of the present invention may be characterized byhaving the usual activity of at least one of the X and Y entitiesforming the fusion or it may be further characterized by having abiological activity greater than simply the additive functions of X andY. This enhancement of activity provides an enhanced therapeuticeffects, thus allowing to reduce dosing regimens, improve compliance andmaintenance therapy, to reduce emergency situations and to improvequality of life. In certain cases, the fusion molecule of the presentinvention may also unexpectedly provide an activity different from thatexpected by the presence of X or Y. For example, one specific unexpectedactivity highlighted in connection with this invention is the ability ofIL-2/IL-18 (IL-2/proIL-18 or IL-2/mature IL-18) and IL-7/IL-2 fusions toactivate the maturation of dendritic cells, for example for the purposeof enhancing a nonspecific immune response against tumor or viralantigens. Another activity discovered for the IL-2/IL-18 fusion(IL-2/proIL-18 or IL-2/mature IL-18) is to activate NKT cells, e.g. forthe purpose of enhancing a nonspecific immune response against tumor orviral antigens. Another unexpected effect discovered in connection withthis invention is the limited cytotoxicity (AICD activity) provided byIL-2/IL-18 (IL-2/proIL-18 or IL-2/mature IL-18) and IL-7/IL-2 fusions ascompared upon administration of the individual cytokine(s) in a givenorganism, which can be used e.g. for reducing cytotoxic side effects.

Further included in the scope of the present invention are novel peptidefragments of the fusion proteins of the invention, and especially ofthose recited in any of SEQ ID NO: 1-19. As used herein, a fragmentcomprises at least 8, 15, 20, 50 or more contiguous amino acid residuesfrom the fusion proteins disclosed herein. Such fragments can be chosenbased on their ability to retain one or more of the therapeutic and/orbiological activities of the fusion protein or could be chosen for theirability to perform a function, e.g. to bind a substrate or to act as animmunogen. Suitable peptide fragments are typically those comprising adomain or motif of the fusion protein containing novel immunogenicstructures. Predicted immunogenic sites are readily identifiable bycomputer programs well known and readily available to those of skill inthe art. Particularly important are peptide fragments overlapping thefusion site between the X and Y entities. Peptide fragments of thefusion protein of the invention can also be synthesized using knownprotein synthesis methods.

The present invention also provides a nucleic acid molecule encoding thefusion protein of the invention.

Within the context of the present invention, the term “nucleic acid” and“polynucleotide” are used interchangeably and define a polymer ofnucleotides of any length, either deoxyribonucleotide (DNA) molecules(e.g., cDNA or genomic DNA) and ribonucleotide (RNA) molecules (e.g.,mRNA) and analogs of the DNA or RNA generated using nucleotide analogs(see U.S. Pat. No. 5,525,711, U.S. Pat. No. 4,711,955 or EPA 302 175 asexamples of nucleotide analogs). If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may also be interrupted bynon-nucleotide elements. The nucleic acid molecule may be furthermodified after polymerization, such as by conjugation with a labelingcomponent. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded, but preferably is double-stranded DNA. Single-strandednucleic acids can be the coding strand (sense strand) or the non-codingstrand (anti-sense strand).

The nucleic acid molecules of the invention include, but are not limitedto, the sequence encoding the fusion protein alone, but may compriseadditional non-coding sequences, for example introns and non-coding 5′and 3′ sequences that play a role in transcription, mRNA processing(including splicing and polyadenylation signals), ribosome binding andmRNA stability. For example, the nucleic acid molecule of the inventioncan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank (i.e. sequences locatedat the 5′ and 3′ ends) or are present within the genomic DNA encoding Xand/or Y entities.

According to a preferred embodiment, the present invention providesnucleic acid molecules which comprise, or alternatively consistessentially of, or alternatively consist of a nucleotide sequenceencoding all or part of an amino acid sequence encoding a fusion proteinwhich is at least about 70%, at least about 75%, at least about 80%, atleast about 90%, at least about 95%, preferably at least about 97%, morepreferably at least about 99% homologous or even more preferably 100%homologous to any of the amino acid sequences shown in SEQ ID NO: 1-19.

In another embodiment, a nucleic acid molecule of the inventioncomprises a nucleic acid molecule which is a complement of all or partof a nucleotide sequence encoding the fusion protein shown in any of SEQID NO: 1-19. A nucleic acid molecule which is complementary to thenucleotide sequence of the present invention is one which issufficiently complementary such that it can hybridize to thefusion-encoding nucleotide sequence under stringent conditions, therebyforming a stable duplex. Such stringent conditions are known to thoseskilled in the art. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6 times sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2 times SSC, 0.1% SDS at 50-65° C. In one embodiment, theinvention pertains to antisense nucleic acid to the nucleic acidmolecules of the invention. The antisense nucleic acid can becomplementary to an entire coding strand, or to only a portion thereof.

In still another embodiment, the invention encompasses variants of theabove-described nucleic acid molecules of the invention, e.g. thatencode variants of the fusion proteins that are described above. Thevariation(s) encompassed by the present invention can be created byintroducing one or more nucleotide substitutions, additions and/ordeletions into the nucleotide sequence by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Followingmutagenesis, the variant nucleic acid molecule can be expressedrecombinantly as described herein and the activity of the resultingprotein can be determined using, for example, assays described herein.Alternatively, the nucleic acid molecule of the invention can be alteredto provide preferential codon usage for a specific host cell (forexample E. coli; Wada et al., 1992, Nucleic Acids Res. 20, 2111-2118).The invention further encompasses nucleic acid molecules that differ dueto the degeneracy of the genetic code and thus encode for example thesame fusion protein as any of those shown in SEQ ID NO: 1-19.

Another embodiment of the invention pertains to fragments of the nucleicacid molecule of the invention, e.g. restriction endonuclease andPCR-generated fragments. Such fragments can be used as probes, primersor fragments encoding an immunogenic portion of the fusion protein.

The nucleic acid molecules of the present invention can be generatedusing the sequence information provided herein. The nucleic acidencoding each of the X and Y entities can be cloned or amplified usingcDNA or, alternatively, genomic DNA, as a template and appropriateprobes or oligonucleotide primers according to standard molecularbiology techniques (e.g., as described in Sambrook, et al. “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001) or standard PCR amplification techniquesbased on sequence data accessible in the art (such as those providedabove in connection with the fusion proteins of the invention or thoseprovided in the Examples part). Fusing of the X sequence to the Ysequence may be accomplished as described in the Examples below or byconventional techniques. For example, the X and Y-encoding sequences canbe ligated together in-frame either directly or through a sequenceencoding a peptide linker. The X-encoding sequence can also be inserteddirectly into a vector which contains the Y-encoding sequence, or viceversa. Alternatively, PCR amplification of the X and Y-encodingsequences can be carried out using primers which give rise tocomplementary overhangs which can subsequently be annealed andre-amplified to generate a fusion gene sequence.

Also provided by the present invention is a vector containing thenucleic acid molecule of the invention.

The term “vector” as used herein refers to both expression andnonexpression vectors and includes viral as well as nonviral vectors,including autonomous self-replicating circular plasmids. Where arecombinant microorganism or cell culture is described as hosting an“expression vector,” this includes both extrachromosomal circular DNAand DNA that has been incorporated into the host chromosome(s).Preferred vectors of the invention are expression vectors. An expressionvector contains multiple genetic elements positionally and sequentiallyoriented, i.e., operatively linked with other necessary elements suchthat nucleic acid molecule in the vector encoding the fusion proteins ofthe invention can be transcribed, and when necessary, translated in thehost cells.

Any type of vector can be used in the context of the present invention,whether of plasmid or viral origin, whether it is an integrating ornonintegrating vector. Such vectors are commercially available ordescribed in the literature. Particularly important in the context ofthe invention are vectors for use in gene therapy (i.e. which arecapable of delivering the nucleic acid molecule to a target cell) aswell as expression vectors for use in recombinant techniques (i.e. whichare capable for example of expressing the nucleic acid molecule of theinvention in cultured host cells).

The vectors of the invention can function in prokaryotic or eukaryoticcells or in both (shuttle vectors). Suitable vectors include withoutlimitation vectors derived from bacterial plasmids, bacteriophages,yeast episomes, artificial chromosomes, such as BAC, PAC, YAC, or MAC,and vectors derived from viruses such as baculoviruses, papovaviruses(e.g. SV40), herpes viruses, adenoviruses, adenovirus-associated viruses(AAV), poxviruses, foamy viruses, and retroviruses. Vectors may also bederived from combinations of these sources such as those derived fromplasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.Viral vectors can be replication-competent, conditionally replicative orreplication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Examples of suitable plasmids include but are not limited to thosederived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript(Stratagene), p Poly (Lathe et al., 1987, Gene 57, 193-201), pTrc (Amannet al., 1988, Gene 69, 301-315) and pET 11d (Studier et al., 1990, GeneExpression Technology: Methods in Enzymology 185, 60-89). It is wellknown that the four of the plasmid can affect the expression efficiency,and it is preferable that a large fraction of the vector be insupercoiled form. Examples of vectors for expression in yeast (e.g. S.cerevisiae) include pYepSec1 (Baldari et al., 1987, EMBO J. 6, 229-234),pMFa (Kujan et al., 1982, Cell 30, 933-943), pJRY88 (Schultz et al.,1987, Gene 54, 113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). The vectors of the invention can also be derived frombaculoviruses to be expressed in cultured insect cells (e.g. Sf 9cells).

According to a preferred embodiment of the invention, the nucleic acidmolecules described herein are expressed by using mammalian expressionvectors. Examples of mammalian expression vectors include pREP4, pCEP4(Invitrogene), pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840) andpMT2PC (Kaufman et al., 1987, EMBO J. 6, 187-195). The expressionvectors listed herein are provided by way of example only of somewell-known vectors available to those of ordinary skill in the art. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance, propagation or expression of the nucleic acidmolecules described herein.

Moreover, the vector of the present invention may also comprise a markergene in order to select or to identify the transfected cells (e.g. bycomplementation of a cell auxotrophy or by antibiotic resistance),stabilising elements (e.g. cer sequence; Summers and Sherrat, 1984, Cell36, 1097-1103), integrative elements (e.g. LTR viral sequences andtransposons) as well as elements providing a self-replicating functionand enabling the vector to be stably maintained in cells, independentlyof the copy number of the vector in the cell. Markers includetetracycline or ampicillin-resistance genes for prokaryotic host cellsand dihydrofolate reductase or neomycin resistance for eukaryotic hostcells. However, any marker that provides selection for a phenotypictrait will be effective. The self-replicating function may be providedby using a viral origin of replication and providing one or more viralreplication factors that are required for replication mediated by thatparticular viral origin (WO 95/32299). Origins of replication and anyreplication factors may be obtained from a variety of viruses, includingEpstein-Barr virus (EBV), human and bovine papilloma viruses andpapovavirus BK.

Particularly preferred vectors of the present invention are viralvectors and especially adenoviral vectors, which have a number ofwell-documented advantages as vectors for gene therapy. The adenoviralgenome consists of a linear double-stranded DNA molecule ofapproximately 36 kb carrying more than about thirty genes necessary tocomplete the viral cycle. The early genes are divided into 4 regions (E1to E4) that are essential for viral replication (Pettersson and Roberts,1986, In Cancer Cells (Vol 4): DNA Tumor Viruses, Botchan and GlodzickerSharp Eds pp 37-47, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.; Halbert et al., 1985, J. Virol. 56, 250-257) with the exception ofthe E3 region, which is believed dispensable for viral replication basedon the observation that naturally-occurring mutants or hybrid virusesdeleted within the E3 region still replicate like wild-type viruses incultured cells (Kelly and Lewis, 1973, J. Virol. 12, 643-652). The E1gene products encode proteins responsible for the regulation oftranscription of the viral genome. The E2 gene products are required forinitiation and chain elongation in viral DNA synthesis. The proteinsencoded by the E3 prevent cytolysis by cytotoxic T cells and tumornecrosis factor (Wold and Gooding, 1991, Virology 184, 1-8). Theproteins encoded by the E4 region are involved in DNA replication, lategene expression and splicing and host cell shut off (Halbert et al.,1985, J. Virol. 56, 250-257). The late genes (L1 to L5) encode in theirmajority the structural proteins constituting the viral capsid. Theyoverlap at least in part with the early transcription units and aretranscribed from a unique promoter (MLP for Major Late Promoter). Inaddition, the adenoviral genome carries at both extremities cis-acting5′ and 3′ ITRs (Inverted Terminal Repeat) and the encapsidation region,both essential for DNA replication. The ITRs harbor origins of DNAreplication whereas the encapsidation region is required for thepackaging of adenoviral DNA into infectious particles.

As used herein, the term “adenoviral vector” encompasses vector DNA aswell as viral particles generated thereof by conventional technologies.

The adenoviral vectors for use in accordance with the present invention,preferably infects human cells. It can be derived from any human oranimal source, in particular canine (e.g. CAV-1 or CAV-2; Genbank refCAV1GENOM and CAV77082 respectively), avian (Genbank ref AAVEDSDNA),bovine (such as BAV3; Seshidhar Reddy et al., 1998, J. Virol. 72,1394-1402), murine (Genbank ref ADRMUSMAV1), ovine, feline, porcine orsimian adenovirus or alternatively from a hybrid thereof. Any serotypecan be employed from the adenovirus serotypes 1 through 51. Forinstance, an adenovirus can be of subgroup A (e.g. serotypes 12, 18, and31), subgroup B (e.g. serotypes 3, 7, 11, 14, 16, 21, 34, and 35),subgroup C (e.g. serotypes 1, 2, 5, and 6), subgroup D (e.g. serotypes8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroupE (serotype 4), subgroup F (serotypes 40 and 41), or any otheradenoviral serotype. However, the human adenoviruses of the B or Csub-group are preferred and especially adenoviruses 2 (Ad2), 5 (Ad5) and35 (Ad35). Generally speaking, adenoviral stocks that can be employed asa source of the cited adenovirus are currently available from theAmerican Type Culture Collection (ATCC, Rockville, Md.), or from anyother source. Moreover, such adenoviruses have been the subject ofnumerous publications describing their sequence, organization andbiology, allowing the artisan to apply them. Adenoviral vectors, methodsof producing adenoviral vectors, and methods of using adenoviral vectorsare disclosed, for example in U.S. Pat. No. 6,133,028 and U.S. Pat. No.6,040,174, U.S. Pat. No. 6,110,735, U.S. Pat. No. 6,399,587, WO 00/50573and EP 1016711 for group C adenoviral vectors and for example in U.S.Pat. No. 6,492,169 and WO 02/40665 for non-group C adenoviral vectors.

In one embodiment, the adenoviral vector of the present invention isreplication-competent. The term “replication-competent” as used hereinrefers to an adenoviral vector capable of replicating in a host cell inthe absence of any trans-complementation. In the context of the presentinvention, this term also encompasses replication-selective orconditionally-replicative adenoviral vectors which are engineered toreplicate better or selectively in cancer or hyperproliferative hostcells. Examples of such replication-competent adenoviral vectors arewell known in the art and readily available to those skill in the art(see, for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11,2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany etal., 2000, Nature Biotechnology 18, 723-727).

Replication-competent adenoviral vectors according to the invention canbe a wild-type adenovirus genome or can be derived therefrom byintroducing modifications into the viral genome, e.g., for the purposeof generating a conditionally-replicative adenoviral vector. Suchmodification(s) include the deletion, insertion and/or mutation of oneor more nucleotide(s) in the coding sequences and/or the regulatorysequences. Preferred modifications are those that render saidreplication-competent adenoviral vector dependent on cellular activitiesspecifically present in a tumor or cancerous cell. In this regard, viralgene(s) that become dispensable in tumor cells, such as the genesresponsible for activating the cell cycle through p53 or Rb binding, canbe completely or partially deleted or mutated. By way of illustration,such conditionally-replicative adenoviral vectors can be engineered bythe complete deletion of the adenoviral MB gene encoding the 55 kDaprotein or the complete deletion of the MB region to abrogate p53binding (see for example U.S. Pat. No. 5,801,029 and U.S. Pat. No.5,846,945). This prevents the virus from inactivating tumor suppressionin normal cells, which means that the virus cannot replicate. However,the virus will replicate and lyse cells that have shut off p53 or Rbexpression through oncogenic transformation. As another example, thecomplete deletion of the E1A region makes the adenoviral vectordependent on intrinsic or IL-6-induced E1A-like activities. Optionally,an inactivating mutation may also be introduced in the E1A region toabrogate binding to the Rb. Rb defective mutation/deletion is preferablyintroduced within the E1A CR1 and/or CR2 domain (see for exampleWO00/24408). In a second strategy optionally or in combination to thefirst approach, native viral promoters controlling transcription of theviral genes can be replaced with tissue or tumor-specific promoters. Byway of illustration, regulation of the E1A and/or the E1B genes can beplaced under the control of a tumor-specific promoter such as the PSA,the kallikrein, the probasin, the AFP, the a-fetoprotein or thetelomerase reverse transcriptase (TERT) promoter (see for example U.S.Pat. No. 5,998,205, WO 99/25860, U.S. Pat. No. 5,698,443 and WO00/46355) or a cell-cycle specific promoter such as E2F-1 promoter(WO00/15820 and WO01/36650). Particularly preferred in this context isthe examplary vector designated ONYX-411 which combines a Rb defectivedeletion of 8 amino acid residues within the MA CR2 domain and the useof E2F-1 promoter to control expression of both the E1A and E4 viralgenes.

According to another and preferred embodiment, the adenoviral vector ofthe invention is replication-defective. Replication-defective adenoviralvectors are known in the art and can be defined as being deficient inone or more regions of the adenoviral genome that are essential to theviral replication (e.g., E1, E2 or E4 or combination thereof), and thusunable to propagate in the absence of trans-complementation (e.g.,provided by either complementing cells or a helper virus). Thereplication-defective phenotype is obtained by introducing modificationsin the viral genome to abrogate the function of one or more viralgene(s) essential to the viral replication. Preferredreplication-defective vectors are E1-deleted, and thus defective in E1function. Such E1-deleted adenoviral vectors include those described inU.S. Pat. No. 6,063,622; U.S. Pat. No. 6,093,567; WO 94/28152; WO98/55639 and EP 974 668 (the disclosures of all of these publicationsare hereby incorporated herein by reference). A preferred E1 deletioncovers approximately the nucleotides (nt) 459 to 3328 or 459 to 3510, byreference to the sequence of the human adenovirus type 5 (disclosed inthe GeneBank under the accession number M 73260 and in Chroboczek etal., 1992, Virol. 186, 280-285).

Furthermore, the adenoviral backbone of the vector may comprisemodifications (e.g. deletions, insertions or mutations) in additionalviral region(s), to abolish the residual synthesis of the viral antigensand/or to improve long-term expression of the nucleic acid molecules inthe transduced cells (see for example WO 94/28152; Lusky et al., 1998,J. Virol 72, 2022-2032; Yeh et al., 1997, FASEB J. 11, 615-623). In thiscontext, the present invention contemplates the use of adenoviralvectors lacking E1, or E1 and E2, or E1 and E3, or E1 and E4, or E1 andE2 and E3, or E1 and E2 and E4, or E1 and E3 and E4, or E1 and E2 and E3and E4 functions. An adenoviral vector defective for E2 function may bedeleted of all or part of the E2 region (preferably within the E2A oralternatively within the E2B or within both the E2A and the E2B regions)or comprises one or more mutations, such as the thermosensitive mutationof the DBP (DNA Binding Protein) encoding gene (Ensinger et al., J.Virol. 10 (1972), 328-339). The adenoviral vector may also be deleted ofall or part of the E4 region (see, for example, EP 974 668 and WO00/12741). An examplary E4 deletion covers approximately the nucleotidesfrom position 32994 to position 34998, by reference to the sequence ofthe human adenovirus type 5. In addition, deletions within thenon-essential E3 region (e.g. from Ad5 position 28597 to position 30469)may increase the cloning capacity, but it may be advantageous to retainthe E3 sequences coding for gp19k, 14.7K and/or RID allowing to escapethe host immune system (Gooding et al., 1990, Critical Review ofImmunology 10, 53-71) and inflammatory reactions (EP 00 440 267.3). Itis also conceivable to employ a minimal (or gutless) adenoviral vectorwhich lacks all functional genes including early (E1, E2, E3 and E4) andlate genes (L1, L2, L3, L4 and L5) with the exception of cis-actingsequences (see for example Kovesdi et al., 1997, Current Opinion inBiotechnology 8, 583-589; Yeh and Perricaudet, 1997, FASEB 11, 615-623;WO 94/12649; and WO 94/28152). The replication-deficient adenoviralvector may be readily engineered by one skilled in the art, taking intoconsideration the required minimum sequences, and is not limited tothese exemplary embodiments.

The nucleic acid molecule of the present invention can be inserted inany location of the adenoviral genome, with the exception of thecis-acting sequences. Preferably, it is inserted in replacement of adeleted region (E1, E3 and/or E4), with a special preference for thedeleted E1 region. In addition, the expression cassette may bepositioned in sense or antisense orientation relative to the naturaltranscriptional direction of the region in question.

A retroviral vector is also suitable in the context of the presentinvention. Retroviruses are a class of integrative viruses whichreplicate using a virus-encoded reverse transcriptase, to replicate theviral RNA genome into double stranded DNA which is integrated intochromosomal DNA of the infected cells. The numerous vectors described inthe literature may be used within the framework of the present inventionand especially those derived from murine leukemia viruses, especiallyMoloney (Gilboa et al., 1988, Adv. Exp. Med. Biol. 241, 29) or Friend'sFB29 strains (WO 95/01447). Generally, a retroviral vector is deleted ofall or part of the viral genes gag, pol and env and retains 5′ and 3′LTRs and an encapsidation sequence. These elements may be modified toincrease expression level or stability of the retroviral vector. Suchmodifications include the replacement of the retroviral encapsidationsequence by one of a retrotransposon such as VL30 (U.S. Pat. No.5,747,323). The nucleic acid molecule of the invention can be inserteddownstream of the encapsidation sequence, preferably in oppositedirection relative to the retroviral genome.

A poxyiral vector is also suitable in the context of the presentinvention. Poxviruses are a group of complex enveloped viruses thatdistinguish from the above-mentioned viruses by their large DNA genomeand their cytoplasmic site of replication. The genome of several membersof poxyiridae has been mapped and sequenced. It is a double-stranded DNAof approximately 200 kb coding for about 200 proteins of whichapproximately 100 are involved in virus assembly. In the context of thepresent invention, a poxyiral vector may be obtained from any member ofthe poxyiridae, in particular canarypox, fowlpox and vaccinia virus, thelatter being preferred. Suitable vaccinia viruses include withoutlimitation the Copenhagen strain (Goebel et al., 1990, Virol. 179,247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), theWyeth strain and the modified Ankara (MVA) strain (Antoine et al., 1998,Virol. 244, 365-396). The general conditions for constructing poxviruscomprising a nucleic acid molecule are well known in the art (see forexample EP 83 286; EP 206 920 for Copenhagen vaccinia viruses and Mayret al., 1975, Infection 3, 6-14; Sutter and Moss, 1992, Proc. Natl.Acad. Sci. USA 89, 10847-10851, U.S. Pat. No. 6,440,422 for MVAviruses). The nucleic acid molecule of the present invention ispreferably inserted within the poxyiral genome in a non-essential locus,such as non-coding intergenic regions or any gene for which inactivationor deletion does not significantly impair viral growth and replication.Thymidine kinase gene is particularly appropriate for insertion inCopenhagen vaccinia viruses (Hruby et al., 1983, Proc. Natl. Acad. Sci.USA 80, 3411-3415; Weir et al., 1983, J. Virol. 46, 530-537). As far asMVA is concerned, insertion of the nucleic acid molecule can beperformed in any of the excisions I to VII, and preferably in excision Hor III (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038; Sutter et al.,1994, Vaccine 12, 1032-1040) or in D4R locus. For fowlpox virus,although insertion within the thymidine kinase gene may be considered,the nucleic acid molecule is preferably introduced into a non-codingintergenic region (see for example EP 314 569 and U.S. Pat. No.5,180,675). One may also envisage insertion in an essential viral locusprovided that the defective function be supplied in trans, via a helpervirus or by expression in the producer cell line. Suitable poxyiralvectors can be readily generated from wild type poxviruses available inrecognized collections such as ATCC (fowlpox ATCC VR-251, monkey poxATCC VR-267, swine pox ATCC VR-363, canarypox ATCC VR-111, cowpox ATCCVR-302) or ICTV (Canberra, Australia) (Copenhagen virus code58.1.1.0.001; GenBank accession number M35027).

According to a preferred embodiment, the vectors of the inventioncomprise the nucleic acid molecule of the invention in a form suitablefor its expression in a host cell or organism, which means that thenucleic acid molecule is placed under the control of one or moreregulatory sequences, selected on the basis of the vector type and/orhost cell, which is operatively linked to the nucleic acid molecule tobe expressed. As used herein, the term “regulatory sequence” refers toany sequence that allows, contributes or modulates the functionalregulation of the nucleic acid molecule, including replication,duplication, transcription, splicing, translation, stability and/ortransport of the nucleic acid or one of its derivative (i.e. mRNA) intothe host cell or organism. In the context of the invention, this termencompasses promoters, enhancers and other expression control elements(e.g., polyadenylation signals and elements that affect mRNA stability).“Operably linked” is intended to mean that the nucleic acid molecule ofinterest is linked to the regulatory sequence(s) in a manner whichallows for expression of the nucleic acid molecule (e.g., in a host cellor organism). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, etc.

Regulatory sequences include promoters which direct constitutiveexpression of a nucleic acid molecule in many types of host cell andthose which direct expression of the nucleotide sequence only in certainhost cells (e.g., tissue-specific regulatory sequences) or in responseto specific events or exogenous factors (e.g. by temperature, nutrientadditive, hormone or other ligand).

Suitable regulatory sequences useful in the context of the presentinvention include, but are not limited to, the left promoter frombacteriophage lambda, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the cytomegalovirus (CMV) immediateearly promoter or enhancer (Boshart et al., 1985, Cell 41, 521-530), theadenovirus early and late promoters, the phosphoglycero kinase (PGK)promoter (Hitzeman et al., 1983, Science 219, 620-625; Adra et al.,1987, Gene 60, 65-74), the thymidine kinase (TK) promoter of herpessimplex virus (HSV)-1 and retroviral long-terminal repeats (e.g. MoMuLVand Rous sarcoma virus (RSV) LTRs). Suitable promoters useful to driveexpression of the nucleic acid molecule of the invention in a poxyiralvector include the 7.5K, H5R, TK, p28, p11 or K1L promoters of vacciniavirus. Alternatively, one may use a synthetic promoter such as thosedescribed in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097),Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar andBoyle (1990, Virology 179, 151-158) as well as chimeric promotersbetween early and late poxyiral promoters.

Inducible promoters are regulated by exogenously supplied compounds, andinclude, without limitation, the zinc-inducible metallothionein (MT)promoter (Mc Ivor et al., 1987, Mol. Cell. Biol. 7, 838-848), thedexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter,the T7 polymerase promoter system (WO 98/10088), the ecdysone insectpromoter (No et al., 1996, Proc. Natl. Acad. Sci. USA 93, 3346-3351),the tetracycline-repressible promoter (Gossen et al., 1992, Proc. Natl.Acad. Sci. USA 89, 5547-5551), the tetracycline-inducible promoter (Kimet al., 1995, J. Virol. 69, 2565-2573), the RU486-inducible promoter(Wang et al., 1997, Nat. Biotech. 15, 239-243 and Wang et al., 1997,Gene Ther. 4, 432-441) and the rapamycin-inducible promoter (Magari etal., 1997, J. Clin. Invest. 100, 2865-2872).

The regulatory sequences in use in the context of the present inventioncan also be tissue-specific to drive expression of the nucleic acidmolecule in the tissues where therapeutic benefit is desired. Examplaryliver-specific regulatory sequences include but are not limited to thoseof HMG-CoA reductase (Luskey, 1987, Mol. Cell. Biol. 7, 1881-1893);sterol regulatory element 1 (SRE-1; Smith et al., 1990, J. Biol. Chem.265, 2306-2310); albumin (Pinkert et al., 1987, Genes Dev. 1, 268-277);phosphoenol pyruvate carboxy kinase (PEPCK) (Eisenberger et al., 1992,Mol. Cell. Biol. 12, 1396-1403); human C-reactive protein (CRP) (Li etal., 1990, J. Biol. Chem. 265, 4136-4142); human glucokinase (Tanizawaet al., 1992, Mol. Endocrinology. 6, 1070-1081); cholesterol 7-alphahydroylase (CYP-7) (Lee et al., 1994, J. Biol. Chem. 269, 14681-14689);alpha-1 antitrypsin (Ciliberto et al., 1985, Cell 41, 531-540);insulin-like growth factor binding protein (IGFBP-1) (Babajko et al.,1993, Biochem Biophys. Res. Comm. 196, 480-486); human transferrin(Mendelzon et al., 1990, Nucl. Acids Res. 18, 5717-5721); collagen typeI (Houglum et al., 1994, J. Clin. Invest. 94, 808-814) and FIX (U.S.Pat. No. 5,814,716) genes. Examplary prostate-specific regulatorysequences include but are not limited to those of the prostatic acidphosphatase (PAP) (Balms et al., 1994, Biochim. Biophys. Acta. 1217,188-194); prostatic secretory protein 94 (PSP 94) (Nolet et al., 1991,Biochim. Biophys. Acta 1089, 247-249); prostate specific antigen complex(Kasper et al., 1993, J. Steroid Biochem. Mol. Biol. 47, 127-135); humanglandular kallikrein (hgt-1) (Lilja et al., 1993, World J. Urology 11,188-191) genes. Examplary pancreas-specific regulatory sequences includebut are not limited to those of pancreatitis associated protein (PAP)promoter (Dusetti et al., 1993, J. Biol. Chem. 268, 14470-14475);elastase 1 transcriptional enhancer (Kruse et al., 1993, Genes andDevelopment 7, 774-786); pancreas specific amylase and elastaseenhancer/promoter (Wu et al., 1991, Mol. Cell. Biol. 11, 4423-4430;Keller et al., 1990, Genes & Dev. 4, 1316-1321); pancreatic cholesterolesterase gene promoter (Fontaine et al., 1991, Biochemistry 30,7008-7014) and the insulin gene promoter (Edlund et al., 1985, Science230, 912-916). Examplary neuron-specific regulatory sequences includebut are not limited to neuron-specific enolase (NSE) (Forss-Petter etal., 1990, Neuron 5, 187-197) and the neurofilament (Byrne and Ruddle,1989, Proc. Natl. Acad. Sci. USA 86, 5473-5477) gene promoters.Examplary regulatory sequences for expression in the brain include butare not limited to the neurofilament heavy chain (NF-H) promoter(Schwartz et al., 1994, J. Biol. Chem. 269, 13444-13450). Examplarylymphoid-specific regulatory sequences include but are not limited tothe human CGL1/granzyme B promoter (Hanson et al., 1991, J. Biol. Chem.266, 24433-24438); terminal deoxy transferase (TdT), lymphocyte specifictyrosine protein kinase (p561ck) promoters (Lo et al., 1991, Mol. Cell.Biol. 11, 5229-5243); the human CD2 promoter/enhancer (Lake et al.,1990, EMBO J. 9, 3129-3136), the human NK and T cell specific activation(NKG5) (Houchins et al., 1993, Immunogenetics 37, 102-107), T cellreceptor (Winoto and Baltimore, 1989, EMBO J. 8, 729-733) andimmunoglobulin (Banerji et al., 1983, Cell 33, 729-740; Queen andBaltimore, 1983, Cell 33, 741-748) promoters. Examplary colon-specificregulatory sequences include but are not limited to pp 60c-src tyrosinekinase (Talamonti et al., 1993, J. Clin. Invest 91, 53-60);organ-specific neoantigens (OSNs), mw 40 kDa (p40) (Ilantzis et al.,1993, Microbiol. Immunol. 37, 119-128); and colon specific antigen-Ppromoter (Sharkey et al., 1994, Cancer 73, 864-877) promoters. Examplaryregulatory sequences for expression in mammary gland and breast cellsinclude but are not limited to the human alpha-lactalbumin (Thean etal., 1990, British J. Cancer. 61, 773-775) and milk whey (U.S. Pat. No.4,873,316) promoters. Examplary muscle-specific regulatory sequencesinclude but are not limited to SM22 (WO 98/15575; WO 97/35974), thedesmin (WO 96/26284), mitochondrial creatine kinase (MCK) promoters, andthe chimeric promoter disclosed in EP 1310561. Exemplary lung-specificregulatory sequences include but are not limited to the CFTR andsurfactant promoters.

Additional promoters suitable for use in this invention can be takenfrom genes that are preferentially expressed in proliferative tumorcells. Such genes can be identified for example by display andcomparative genomic hybridization (see for example U.S. Pat. Nos.5,759,776 and 5,776,683). Examplary tumor specific promoters include butare not limited to the promoters of the MUC-1 gene overexpressed inbreast and prostate cancers (Chen et al., 1995, J. Clin. Invest. 96,2775-2782), of the Carcinoma Embryonic Antigen (CEA)-encoding geneoverexpressed in colon cancers (Schrewe et al., 1990, Mol. Cell. Biol.10, 2738-2748), of the ERB-2 encoding gene overexpressed in breast andpancreas cancers (Harris et al., 1994, Gene Therapy 1, 170-175), of thealpha-foetoprotein gene overexpressed in liver cancers (Kanai et al.,1997, Cancer Res. 57, 461-465), of the telomerase reverse transcriptase(TERT) (WO99/27113, WO 02/053760 and Horikawa et al., 1999, Cancer Res.59, 826), hypoxia-responsive element (HRE), autocrine motility factorreceptor, L plasmin and hexokinase II.

Those skilled in the art will appreciate that the regulatory elementscontrolling the expression of the nucleic acid molecule of the inventionmay further comprise additional elements for proper initiation,regulation and/or termination of transcription and translation into thehost cell or organism. Such additional elements include but are notlimited to non coding exon/intron sequences, transport sequences,secretion signal sequences, nuclear localization signal sequences, IRES,polyA transcription termination sequences, tripartite leader sequences,sequences involved in replication or integration. Illustrative examplesof introns suitable in the context of the invention include thoseisolated from the genes encoding alpha or beta globin (i.e. the secondintron of the rabbit beta globin gene; Green et al., 1988, Nucleic AcidsRes. 16, 369; Karasuyama et al., 1988, Eur. J. Immunol. 18, 97-104),ovalbumin, apolipoprotein, immunoglobulin, factor IX, and factor VIII,the SV40 16S/19S intron (Okayma and Berg, 1983, Mol. Cell. Biol. 3,280-289) as well as synthetic introns such as the intron present in thepCI vector (Promega Corp, pCI mammalian expression vector E1731) made ofthe human beta globin donor fused to the mouse immunoglobin acceptor or.Where secretion of the fusion protein is desired, appropriate secretionsignals are incorporated into the vector. The signal sequence can beendogenous to the fusion protein (e.g. endogenous to the X or Y entity)or heterologous to both X and Y entities involved in the fusion protein.The person of ordinary skill in the art would be aware of the numerousregulatory sequences that are useful in expression vectors.

A preferred embodiment of the invention is directed to a E1- andE3-deleted replication-defective adenoviral vector comprising thenucleic acid molecule of the invention inserted in replacement of the E1region and placed under the control of the CMV promoter.

In addition, the vector of the invention can further comprise one ormore transgenes (i.e. a gene of interest to be expressed together withthe nucleic acid molecule of the invention in a host cell or organism).Desirably, the expression of the transgene has a therapeutic orprotective activity to the disease or illness condition for which thevector of the present invention is being given. Suitable transgenesinclude without limitation genes encoding (i) tumor proliferationinhibitors and/or (ii) at least one specific antigen against which animmune response is desired. In a preferred form of the presentinvention, the transgene product and the fusion protein actsynergistically in the induction of immune responses or in providing atherapeutic (e.g. antitumoral) benefit. Accordingly, such combinationsare not only suitable for immunoprophylaxis of diseases, butsurprisingly also for immunotherapy of diseases such as viral, bacterialor parasitic infections, and also chronic disorders such as cancers.

Tumor proliferation inhibitors act by directly inhibiting cell growth,or killing the tumor cells. Representative examples of tumorproliferation inhibitors include toxins and suicide genes.Representative examples of toxins include without limitation ricin (Lambet al., 1985, Eur. J. Biochem. 148, 265-270), diphtheria toxin (Twetenet al., 1985, J. Biol. Chem. 260, 10392-10394), cholera toxin (Mekalanoset al., 1983, Nature 306, 551-557; Sanchez and Holmgren, 1989, Proc.Natl. Acad. Sci. USA 86, 481-485), gelonin (Stirpe et al., 1980, J.Biol. Chem. 255, 6947-6953), antiviral protein (Barbieri et al., 1982,Biochem. J. 203, 55-59; Irvin et al., 1980, Arch. Biochem. Biophys. 200,418-425), tritin, Shigella toxin (Calderwood et al., 1987, Proc. Natl.Acad. Sci. USA 84, 4364-4368; Jackson et al., 1987, Microb. Path. 2,147-153) and Pseudomonas exotoxin A (Carroll and Collier, 1987, J. Biol.Chem. 262, 8707-8711).

<<Suicide genes>> can be defined in the context of the present inventionas any gene encoding an expression product able to transform an inactivesubstance (prodrug) into a cytotoxic substance, thereby giving rise tocell death. The gene encoding the TK HSV-1 constitutes the prototypemember of the suicide gene family (Caruso et al., 1993, Proc. Natl.Acad. Sci. USA. 90, 7024-7028; Culver et al., 1992, Science 256,1550-1552). While the TK, polypeptide is non-toxic as such, it catalyzesthe transformation of nucleoside analogs (prodrug) such as acyclovir organciclovir. The transformed nucleosides are incorporated into the DNAchains which are in the process of elongation, cause interruption ofsaid elongation and therefore inhibition of cell division. A largenumber of suicide gene/prodrug combinations are currently available.Those which may more specifically be mentioned are rat cytochrome p450and cyclophosphophamide (Wei et al., 1994, Human Gene Ther. 5, 969-978),Escherichia coli (E. coli) purine nucleoside phosphorylase and6-methylpurine deoxyribonucleoside (Sorscher et al., 1994, Gene Therapy1, 223-238), E. coli guanine phosphoribosyl transferase and6-thioxanthine (Mzoz et al., 1993, Human Gene Ther. 4, 589-595).However, in a preferred embodiment, the vector of the inventioncomprises a suicide gene encoding a polypeptide having a cytosinedeaminase (CDase) or a uracil phosphoribosyl transferase (UPRTase)activity or both CDase and UPRTase activities, which can be used withthe prodrug 5-fluorocytosine (5-FC). Suitable CDase encoding genesinclude but are not limited to the Saccharomyces cerevisiae FCY1 gene(Erbs et al., 1997, Curr. Genet. 31, 1-6; WO 93/01281) and the E. colicodA gene (EP 402 108). Suitable UPRTase encoding genes include but arenot limited to those from E. coli (upp gene; Anderson et al., 1992, Eur.J. Biochem. 204, 51-56), and Saccharomyces cerevisiae (FUR-1 gene; Kernet al., 1990, Gene 88, 149-157). Preferably, the CDase encoding gene isderived from the FCY1 gene and the UPRTase encoding gene is derived fromthe FUR-1 gene. Particularly important is the use of a fusion proteinwhich encodes a two domain enzyme possessing both CDase and UPRTaseactivities (FCU-1) as described in WO 99/54481 (incorporated herein byreference).

Specific antigens are preferably those susceptible to confer an immuneresponse, specific and/or nonspecific, antibody and/or cell-mediated,against a given pathogen (virus, bacterium, fungus or parasite) oragainst a non-self antigen (e.g. a tumor-associated antigen).Preferably, the selected antigen comprises an epitope that binds to, andis presented onto the cell surface by MHC class I proteins.Representative examples of specific antigens include without limitation:

-   -   antigen(s) of the Hepatitis B surface antigen are well known in        the art and include, inter alia, those PreS1, Pars2 S antigens        set forth described in European Patent applications EP 414 374;        EP 304 578, and EP 198 474.    -   Antigens of the Hepatitis C virus including any immunogenic        antigen or fragment thereof selected from the group consisting        of the Core (C), the envelope glycoprotein E1, E2, the        non-structural polypeptide NS2, NS3, NS4 (NS4a and/or NS4b), NS5        (NS5a and/or NS5b) or any combination thereof (e.g. NS3 and NS4,        NS3 and NS4 and NS5b)    -   Antigen(s) of the HIV-1 virus, especially gp120 and gp160 (as        described WO 87/06260).    -   Antigen(s) derived from the Human Papilloma Virus (HPV)        considered to be associated with genital warts (HPV 6 or HPV 11        and others), and cervical cancer (HPV16, HPV18, HPV 31, HPV-33        and others). Particularly important HPV antigens are selected        among the group consisting of E5, E6, E7, L1, and L2 either        individually or in combination (see for example WO 94/00152, WO        94/20137, WO 93/02184, WO 90/10459, and WO 92/16636).        Particularly important in the context of the invention are        membrane anchored forms of non oncogenic variants of the early        HPV-16 E6 and/or E7 antigens (as described in WO 99/03885) that        are particularly suitable to achieve an anti-tumoral effect        against an HPV-associated cancer.    -   antigens from parasites that cause malaria. For example,        preferred antigens from Plasmodia falciparum include RTS (WO        93/10152), and TRAP (WO 90/01496). Other plasmodia antigens that        are likely candidates are P. falciparum. MSP1, AMA1, MSP3, EBA,        GLURP, RAPT, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP,        SALSA, PfEXP1, Pfs25, Pfs28, PFS27125, Pfs16, Pfs48/45, Pfs230        and their analogues in other Plasmodium species.    -   Other suitable antigens include tumour-associated antigens such        as those associated with prostrate, breast, colorectal, lung,        pancreatic, renal, liver, bladder, sarcoma or melanoma cancers.        Exemplary antigens include MAGE 1, 3 and MAGE 4 or other MAGE        antigens (WO 99/40188), PRAME, BAGE, Lage (also known as NY        Eos 1) SAGE and HAGE (WO 99/53061) or GAGE (Robbins and        Kawakami, 1996. Current Opinions in Immunol. 8, pps 628-636).        Other suitable tumor-associated antigens include those known as        prostase, including Prostate specific antigen (PSA), PAP, PSCA,        PSMA. Prostase nucleotide sequence and deduced polypeptide        sequence and homologs are disclosed in Ferguson, et al. (1999,        Proc. Natl. Acad. Sci. USA. 96, 3114-3119) and WO 98/12302 WO        98/20117 and WO 00/04149. Other suitable tumour-associated        antigens include those associated with breast cancer, such as        BRCA-1, BRCA-2 and MUC-1 (see for example WO 92/07000).

The transgene in use in the present invention is placed under thecontrol of appropriate regulatory elements to permit its expression inthe selected host cell or organism in either a constitutive or induciblefashion. The choice of such regulatory elements is within the reach ofthe skilled artisan. It is preferably selected from the group consistingof constitutive, inducible, tumor-specific and tissue-specific promotersas described above in connection with the expression of the fusionprotein of the present invention. In one example, the transgene isplaced under control of the CMV promoter to ensure high levelexpression.

The transgene in use in the present invention can be inserted in anylocation of the vector. According to one alternative, it is placedpreferably not in close proximity of the nucleic acid molecule of theinvention. According to another alternative it can be placed inantisense orientation with respect to the nucleic acid molecule, inorder to avoid transcriptional interference between the two expressioncassettes. For example, in an adenoviral genome, the transgene can beinserted in a different deleted region with respect to the nucleic acidmolecule of the invention (E1, E3 and/or E4) or in the same deletedregion as said nucleic acid molecule but in antisense orientation to oneanother.

Introducing the nucleic acid molecule of the invention into a vectorbackbone can proceed by any genetic engineering strategy appropriate inthe art for any kind of vectors such as by methods described in Sambrooket al. (2001, Molecular Cloning—A Laboratory Manual, Cold Spring HarborLaboratory). Typically, for the introduction of the nucleic acidmolecule into an adenoviral vector, a bacterial plasmid comprising thefusion-encoding nucleic acid molecule is engineered to replace anadenoviral gene required for replication or assembly (e.g. E1) with thesubstitute nucleic acid molecule. The plasmid is then used as a shuttlevector, and combined with a second plasmid containing the complementaryportion of the adenovirus genome, permitting homologous recombination tooccur by virtue of overlapping adenovirus sequences in the two plasmids.The recombination can be done directly in a suitable mammalian host(such as 293 as described in Graham and Prevect, 1991, Methods inMolecular Biology, Vol 7 “Gene Transfer and Expression Protocols”; Ed E.J. Murray, The Human Press Inc, Clinton, N.J.), or else in yeast YACclones or E. coli (as described in WO 96/17070). The completedadenovirus genome is subsequently transfected into mammalian host cellsfor replication and viral encapsidation.

The present invention also encompasses vectors of the invention orparticles thereof that have been modified to allow preferentialtargeting of a particular target cell. A characteristic feature oftargeted vectors/particles of the invention (of both viral and non-viralorigins, such as polymer- and lipid-complexed vectors) is the presenceat their surface of a targeting moiety capable of recognizing andbinding to a cellular and surface-exposed component. Such targetingmoieties include without limitation chemical conjugates, lipids,glycolipids, hormones, sugars, polymers (e.g. PEG, polylysine, PEI andthe like), peptides, polypeptides (for example JTS1 as described in WO94/40958), oligonucleotides, vitamins, antigens, lectins, antibodies andfragments thereof. They are preferably capable of recognizing andbinding to cell-specific markers, tissue-specific markers, cellularreceptors, viral antigens, antigenic epitopes or tumor-associatedmarkers. In this regard, cell targeting of adenoviruses can be carriedout by genetic modification of the viral gene encoding the capsidpolypeptide present on the surface of the virus (e.g. fiber, pentonand/or pIX). Examples of such modifications are described in literature(for example in Wickam et al., 1997, J. Viral. 71, 8221-8229; Amberg etal., 1997, Virol. 227, 239-244; Michael et al., 1995, Gene Therapy 2,660-668; WO 94/10323, EP 02 360204 and WO 02/96939). To illustrate,inserting a sequence coding for EGF within the sequence encoding theadenoviral fiber will allow to target EGF receptor expressing cells. Themodification of poxyiral tropism can also be achieved as described in EP1 146 125. Other methods for cell specific targeting can be achieved bythe chemical conjugation of targeting moieties at the surface of a viralparticle.

In another embodiment, the present invention relates to infectious viralparticles comprising the above-described nucleic acid molecules orvectors of the present invention.

The invention also relates to a process for producing an infectiousviral particle, comprising the steps of:

-   -   (a) introducing the viral vector of the invention into a        suitable cell line,    -   (b) culturing said cell line under suitable conditions so as to        allow the production of said infectious viral particle, and    -   (c) recovering the produced infectious viral particle from the        culture of said cell line, and    -   (d) optionally purifying said recovered infectious viral        particle.

The vector containing the nucleic acid molecule of the invention can beintroduced into an appropriate cell line for propagation or expressionusing well-known techniques readily available to the person of ordinaryskill in the art. These include, but are not limited to, microinjectionof minute amounts of DNA into the nucleus of a cell (Capechi et al.,1980, Cell 22, 479-488), CaPO₄-mediated transfection (Chen and Okayama,1987, Mol. Cell Biol. 7, 2745-2752), DEAE-dextran-mediated transfection,electroporation (Chu et al., 1987, Nucleic Acid Res. 15, 1311-1326),lipofection/liposome fusion (Feigner et al., 1987, Proc. Natl. Acad.Sci. USA 84, 7413-7417), particle bombardment (Yang et al., 1990, Proc.Natl. Acad. Sci. USA 87, 9568-9572), gene guns, transduction, infection(e.g. with an infective viral particle), and other techniques such asthose found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001).

When the vector of the invention is defective, the infectious particlesare usually produced in a complementation cell line or via the use of ahelper virus, which supplies in trans the non functional viral genes.For example, suitable cell lines for complementing adenoviral vectorsinclude the 293 cells (Graham et al., 1997, J. Gen. Virol. 36, 59-72) aswell as the PER-C6 cells (Fallaux et al., 1998, Human Gene Ther. 9,1909-1917) commonly used to complement the E1 function. Other cell lineshave been engineered to complement doubly defective adenoviral vectors(Yeh et al., 1996, J. Virol. 70, 559-565; Krougliak and Graham, 1995,Human Gene Ther. 6, 1575-1586; Wang et al., 1995, Gene Ther. 2, 775-783;Lusky et al., 1998, J. Virol. 72, 2022-2033; WO94/28152 and WO97/04119).The infectious viral particles may be recovered from the culturesupernatant but also from the cells after lysis and optionally arefurther purified according to standard techniques (chromatography,ultracentrifugation in a cesium chloride gradient as described forexample in WO 96/27677, WO 98/00524, WO 98/22588, WO 98/26048, WO00/40702, EP 1016700 and WO 00/50573).

The invention also relates to host cells which comprise the nucleic acidmolecules, vectors or infectious viral particles of the inventiondescribed herein. For the purpose of the invention, the term “host cell”should be understood broadly without any limitation concerningparticular organization in tissue, organ, or isolated cells. Such cellsmay be of a unique type of cells or a group of different types of cellsand encompass cultured cell lines, primary cells and proliferativecells.

Host cells therefore include prokaryotic cells, lower eukaryotic cellssuch as yeast, and other eukaryotic cells such as insect cells, plantand higher eukaryotic cells, such as vertebrate cells and, with aspecial preference, mammalian (e.g. human or non-human) cells. Suitablemammalian cells include but are not limited to hematopoïetic cells(totipotent, stem cells, leukocytes, lymphocytes, monocytes,macrophages, APC, dendritic cells, non-human cells and the like),pulmonary cells, tracheal cells, hepatic cells, epithelial cells,endothelial cells, muscle cells (e.g. skeletal muscle, cardiac muscle orsmooth muscle) or fibroblasts. Preferred host cells include Escherichiacoli, Bacillus, Listeria, Saccharomyces, BHK (baby hamster kidney)cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells(Crandell feline kidney cell line), CV-1 cells (African monkey kidneycell line), COS (e.g., COS-7) cells, chinese hamster ovary (CHO) cells,mouse NIH/3T3 cells, HeLa cells and Vero cells. Host cells alsoencompass complementing cells capable of complementing at least onedefective function of a replication-defective vector of the invention(e.g. adenoviral vector) such as those cited above.

The host cell of the invention can contain more than one nucleic acidmolecule, vector or infectious viral particle of the invention. Furtherit can additionally comprise a vector encoding a transgene, e.g. atransgene as described above. When more than one nucleic acid molecule,vector or infectious viral particle is introduced into a cell, thenucleic acid molecules, vectors or infectious viral particles can beintroduced independently or co-introduced.

Moreover, according to a specific embodiment, the host cell of theinvention can be further encapsulated. Cell encapsulation technology hasbeen previously described (Tresco et al., 1992, ASAJO J. 38, 17-23;Aebischer et al., 1996, Human Gene Ther. 7, 851-860). According to saidspecific embodiment, transfected or infected eukaryotic host cells areencapsulated with compounds which form a microporous membrane and saidencapsulated cells can further be implanted in vivo. Capsules containingthe cells of interest may be prepared employing hollow microporousmembranes (e.g. Akzo Nobel Faser AG, Wuppertal, Germany; Deglon et al.1996, Human Gene Ther. 7, 2135-2146) having a molecular weight cutoffappropriate to permit the free passage of proteins and nutrients betweenthe capsule interior and exterior, while preventing the contact oftransplanted cells with host cells.

Still a further aspect of the present invention is a method forrecombinantly producing the fusion protein, employing the vectors,infectious viral particles and/or host cells of the invention. Themethod for producing the fusion protein comprises introducing a vectoror an infectious viral particle of the invention into a suitable hostcell to produce a transfected or infected host cell, culturing in-vitrosaid transfected or infected host cell under conditions suitable forgrowth of the host cell, and thereafter recovering said fusion proteinfrom said culture, and optionally, purifying said recovered fusionprotein. It is expected that those skilled in the art are knowledgeablein the numerous expression systems available for expression of thefusion proteins of the invention in appropriate host cells.

The host cell of the invention is preferably produced bytransfecting/infecting a host cell with one or more recombinantmolecules, (e.g. a vector of the invention) comprising one or morenucleic acid molecules of the present invention. Recombinant DNAtechnologies can be used to improve expression of the nucleic acidmolecule in the host cell by manipulating, for example, the number ofcopies of the nucleic acid molecule within a host cell, the efficiencywith which the nucleic acid molecule is transcribed, the efficiency withwhich the resultant transcripts are translated, the efficiency ofpost-translational modifications and the use of appropriate selection.Recombinant techniques useful for increasing the expression of nucleicacid molecules of the present invention include, but are not limited to,the use of high-copy number vectors, addition of vector stabilitysequences, substitution or modification of one or more transcriptionalregulatory sequences (e.g., promoters, operators, enhancers),substitution or modification of translational regulatory sequences(e.g., ribosome binding sites, Shine-Dalgamo sequences), modification ofnucleic acid molecule of the present invention to correspond to thecodon usage of the host cell, and deletion of sequences that destabilizetranscripts.

Host cells of the present invention can be cultured in conventionalfermentation bioreactors, flasks, and petri plates. Culturing can becarried out at a temperature, pH and oxygen content appropriate for agiven host cell. No attempts to describe in detail the various methodsknown for the expression of proteins in prokaryote and eukaryote cellswill be made here. In one embodiment, the vector is a plasmid carryingthe fusion-encoding nucleic acid molecule in operative association withappropriate regulatory elements. Preferred host cells in use in themethod of the invention are mammalian cell lines, yeast cells andbacterial cells.

Where the fusion protein is not secreted outside the producing cell orwhere it is not secreted completely, it can be recovered from the cellby standard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. If secreted,it can be recovered directly from the culture medium. The fusion proteincan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, gelelectrophoresis, reverse phase chromatography, size exclusionchromatography, ion exchange chromatography, affinity chromatography,phosphocellulose chromatography, hydrophobic-interaction chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography. The conditions and technology used topurify a particular fusion protein of the invention will depend on thesynthesis method and on factors such as net charge, molecular weight,hydrophobicity, hydrophilicity and will be apparent to those havingskill in the art. It is also understood that depending upon the hostcell used for the recombinant production of the fusion proteinsdescribed herein, the fusion proteins can have various glycosylationpatterns, or may be non-glycosylated (e.g. when produced in bacteria).In addition, the fusion protein may include an initial methionine insome cases as a result of a host-mediated process.

The fusion protein of the invention can be “purified” to the extent thatit is substantially free of cellular material. The level of purificationwill be based on the intended use. The critical feature is that thepreparation allows for the desired function of the fusion protein, evenif in the presence of considerable amounts of other components. In someuses, “substantially free of cellular material” includes preparations ofthe fusion protein having less than about 30% (by dry weight) otherproteins (i.e., contaminating proteins), preferably less than about 20%other proteins, more preferably less than about 10% other proteins, oreven more preferably less than about 5% other proteins. When the fusionprotein is recombinantly produced, it can also be substantially free ofculture medium, i.e., culture medium represents less than about 20% ofthe volume of the protein preparation.

In another aspect, this invention provides a pharmaceutical compositioncomprising an effective amount of the fusion protein, the expressionvector, the infectious viral particle, the host cell of the invention orany combination thereof (also referred herein to “active agents”) andoptionally a pharmaceutically acceptable vehicle. In a special case, thecomposition may comprise two or more active agents, which may differ by(i) the nature of the encoded fusion protein and/or (ii) the nature ofthe regulatory sequence used to express the fusion protein and/or (iii)the additional presence of a transgene and/or (iv) the vector backbone.

The compositions of the present invention may be used to protect ortreat a mammal susceptible to, or suffering from a disease, by means ofadministering said composition by a variety of modes of administrationincluding systemic, topical and localized administration. For systemicadministration, injection is preferred, e.g. subcutaneous, intradermal,intramuscular, intravenous, intraperitoneal, intrathecal, intracardiac(such as transendocardial and pericardial), intratumoral, intravaginal,intrapulmonary, intranasal, intratracheal, intravascular, intraarterial,intracoronary, intracerebroventricular, transdermal (topical) ordirectly into a lymph node. Intramuscular, intradermal, intravenous, orintratumoral administration constitutes the preferred routes forsystemic administration. Alternatively the composition of the presentinvention may be administered via a mucosal route, such as theoral/alimentary, nasal, intratracheal, intravaginal or intra-rectalroute. The preferred mucosal route of administration is via the nasal orintratracheal route.

As used herein the language “pharmaceutically acceptable vehicle” isintended to include any and all carriers, solvents, diluents,excipients, adjuvants, dispersion media, coatings, antibacterial andantifungal agents, and absorption delaying agents, and the like,compatible with pharmaceutical administration.

Suitably, the pharmaceutical composition of the invention comprises acarrier and/or diluent appropriate for its delivering by injection to ahuman or animal organism. Such carrier and/or diluent is non-toxic atthe dosage and concentration employed. It is selected from those usuallyemployed to formulate compositions for parental administration in eitherunit dosage or multi-dose form or for direct infusion by continuous orperiodic infusion. It is preferably isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as provided bysugars, polyalcohols and isotonic saline solutions. Representativeexamples include sterile water, physiological saline (e.g. sodiumchloride), bacteriostatic water, Ringer's solution, glucose orsaccharose solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions (see for example the most current edition ofRemington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott,Williams & Wilkins). The pH of the composition of the invention issuitably adjusted and buffered in order to be appropriate for use inhumans or animals, preferably at a physiological or slightly basic pH(between about pH 8 to about pH 9, with a special preference for pH8.5). Suitable buffers include phosphate buffer (e.g. PBS), bicarbonatebuffer and/or Tris buffer. A particularly preferred composition isformulated in 1M saccharose, 150 mM NaCl, 1 mM MgCl₂, 54 mg/l Tween 80,10 mM Tris pH 8.5. Another preferred composition is formulated in 10mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl. Thesecompositions are stable at −70° C. for at least six months.

The composition of the invention can be in various forms, e.g. in solid(e.g. powder, lyophilized form), or liquid (e.g. aqueous). In the caseof solid compositions, the preferred methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active agent plusany additional desired ingredient from a previously sterile-filteredsolution thereof. Such solutions can, if desired, be stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection.

Nebulised or aerosolised formulations also form part of this invention.Methods of intranasal administration are well known in the art,including the administration of a droplet, spray, or dry powdered formof the composition into the nasopharynx of the individual to be treatedfrom a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer (see forexample WO 95/11664). Enteric formulations such as gastroresistantcapsules and granules for oral administration, suppositories for rectalor vaginal administration also form part of this invention. Fornon-parental administration, the compositions can also includeabsorption enhancers which increase the pore size of the mucosalmembrane. Such absorption enhancers include sodium deoxycholate, sodiumglycocholate, dimethyl-beta-cyclodextrin,lauroyl-1-lysophosphatidylcholine and other substances having structuralsimilarities to the phospholipid domains of the mucosal membrane.

The composition can also contain other pharmaceutically acceptableexcipients for providing desirable pharmaceutical or pharmacodynamicproperties, including for example modifying or maintaining the pH,osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution of the formulation, modifying or maintaining release orabsorption into an the human or animal organism. For example, polymerssuch as polyethylene glycol may be used to obtain desirable propertiesof solubility, stability, half-life and other pharmaceuticallyadvantageous properties (Davis et al., 1978, Enzyme Eng. 4, 169-173;Burnham et al., 1994, Am. J. Hosp. Pharm. 51, 210-218). Representativeexamples of stabilizing components include polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Otherstabilizing components especially suitable in plasmid-based compositionsinclude hyaluronidase (which is thought to destabilize the extracellular matrix of the host cells as described in WO 98/53853),chloroquine, protic compounds such as propylene glycol, polyethyleneglycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or derivativesthereof, aprotic compounds such as dimethylsulfoxide (DMSO),diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane,dimethyl-formamide, dimethylacetamide, tetramethylurea, acetonitrile(see EP 890 362), nuclease inhibitors such as actin G (WO 99/56784) andcationic salts such as magnesium (Mg²⁺) (EP 998 945) and lithium (Li⁺)(WO 01/47563) and any of their derivatives. The amount of cationic saltin the composition of the invention preferably ranges from about 0.1 mMto about 100 mM, and still more preferably from about 0.1 mM to about 10mM. Viscosity enhancing agents include sodium carboxymethylcellulose,sorbitol, and dextran. The composition can also contain substances knownin the art to promote penetration or transport across the blood barrieror membrane of a particular organ (e.g. antibody to transferrinreceptor; Friden et al., 1993, Science 259, 373-377). A gel complex ofpoly-lysine and lactose (Midoux et al., 1993, Nucleic Acid Res. 21,871-878) or poloxamer 407 (Pastore, 1994, Circulation 90, 1-517) can beused to facilitate administration in arterial cells.

The composition of the invention may also comprise one or moreadjuvant(s) suitable for systemic or mucosal application in humans.Representative examples of useful adjuvants include without limitationalum, mineral oil emulsion such as Freunds complete and incomplete,lipopolysaccharide or a derivative thereof (Ribi et al., 1986,Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ.Corp., NY, p407-419), saponins such as QS21 (Sumino et al., 1998, J.Virol. 72, 4931-4939; WO 98/56415), Escin, Digitonin, Gypsophila orChenopodium quinoa saponins. Alternatively the composition of theinvention may be formulated with conventional vaccine vehicles composedof chitosan or other polycationic polymers, polylactide andpolylactide-co-glycolide particles, poly-N-acetyl glucosamine-basedpolymer matrix, particles composed of polysaccharides or chemicallymodified polysaccharides, and lipid-based particles, etc. Thecomposition may also be formulated in the presence of cholesterol toform particulate structures such as liposomes.

The composition may be administered to patients in an amount effective,especially to enhance an immune response in an animal or human organism.As used herein, the term <<effective amount>> refers to an amountsufficient to realize a desired biological effect. For example, aneffective amount for enhancing an immune response could be that amountnecessary to cause activation of the immune system, for instanceresulting in the development of an anti-tumor response in a cancerouspatient (e.g. size reduction or regression of the tumor into which thecomposition has been injected and/or distant tumors). The appropriatedosage may vary depending upon known factors such as the pharmacodynamiccharacteristics of the particular active agent, age, health, and weightof the host organism; the condition(s) to be treated, nature and extentof symptoms, kind of concurrent treatment, frequency of treatment, theneed for prevention or therapy and/or the effect desired. The dosagewill also be calculated dependent upon the particular route ofadministration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by a practitioner, in the light of the relevant circumstances. Forgeneral guidance, a composition based on viral (e.g. adenoviral)particles may be formulated in the form of doses of between 10⁴ and 10¹⁴in (infectious units), advantageously between 10⁵ and 10¹³ iu andpreferably between 10⁶ and 10¹² iu. The titer may be determined byconventional techniques. A composition based on vector plasmids may beformulated in the form of doses of between 1 μg to 100 mg,advantageously between 10 μg and 10 mg and preferably between 100 μg and1 mg. A composition based on proteins may be formulated in the form ofdoses of between 10 ng to 100 mg. A preferred dose is from about 1 μg toabout 10 mg of the therapeutic protein per kg body weight. Theadministration may take place in a single dose or a dose repeated one orseveral times after a certain time interval. In one preferredembodiment, the composition of the present invention is administered byinjection using conventional syringes and needles, or devices designedfor ballistic delivery of solid compositions (WO 99/27961), orneedleless pressure liquid jet device (U.S. Pat. No. 4,596,556; U.S.Pat. No. 5,993,412).

The composition of the invention can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic. In all cases,the composition must be sterile and should be fluid to the extent thateasy syringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. Sterile injectablesolutions can be prepared by incorporating the active agent (e.g., afusion protein or infectious particles) in the required amount with oneor a combination of ingredients enumerated above, followed by filteredsterilization.

The pharmaceutical composition of the invention may be employed inmethods for treating or preventing a variety of diseases and pathologicconditions, including genetic diseases, congenital diseases and acquireddiseases such as infectious diseases (e.g. viral and/or bacterialinfections), cancer, immune deficiency diseases, and autoimmunediseases. Accordingly, the present invention also encompasses the use ofthe fusion protein, vector, infectious viral particle, host cell orcomposition of the invention for the preparation of a drug intended fortreating or preventing such diseases, and especially cancer or aninfectious disease.

The composition of the present invention is particularly intended forthe preventive or curative treatment of disorders, conditions ordiseases associated with cancer. The term “cancer” encompasses anycancerous conditions including diffuse or localized tumors, metastasis,cancerous polyps and preneoplastic lesions (e.g. dysplasies) as well asdiseases which result from unwanted cell proliferation. A variety oftumors may be selected for treatment in accordance with the methodsdescribed herein. In general, solid tumors are preferred. Cancers whichare contemplated in the context of the invention include withoutlimitation glioblastoma, sarcoma, melanomas, mastocytoma, carcinomas aswell as breast cancer, prostate cancer, testicular cancer, ovariancancer, endometrial cancer, cervical cancer (in particular, thoseinduced by a papilloma virus), lung cancer (e.g. lung carcinomasincluding large cell, small cell, squamous and adeno-carcinomas), renalcancer, bladder cancer, liver cancer, colon cancer, anal cancer,pancreatic cancer, stomach cancer, gastrointestinal cancer, cancer ofthe oral cavity, larynx cancer, brain and CNS cancer, skin cancer (e.g.melanoma and non-melanoma), blood cancer (lymphomas, leukemia,especially if they have developed in solid mass), bone cancer,retinoblastoma and thyroid cancer. In one preferred embodiment of theuse of the invention, the composition is administered into or in closeproximity to a solid tumor.

Other pathologic diseases and conditions are also contemplated in thecontext of the invention, especially infectious diseases associated withan infection by a pathogen such as fungi, bacteria, protozoa andviruses. Representative examples of viral pathogens include withoutlimitation human immunodeficiency virus (e.g. HIV-1 or HIV-2), humanherpes viruses (e.g. HSV1 or HSV2), cytomegalovirus, Rotavirus, EpsteinBarr virus (EBV), hepatitis virus (e.g. hepatitis B virus, hepatitis Avirus, hepatitis C virus and hepatitis E virus), varicella-zoster virus(VZV), paramyxoviruses, coronaviruses; respiratory syncytial virus,parainfluenza virus, measles virus, mumps virus, flaviviruses (e.g.Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus), influenza virus, and preferably humanpapilloma viruses (e.g. HPV-6, 11, 16, 18, 31. 33). Representativeexamples of bacterial pathogens include Neisseria (e.g. N. gonorrhea andN. meningitidis); Bordetella (e.g. B. pertussis, B. parapertussis and B.bronchiseptica), Mycobacteria (e.g. M. tuberculosis, M. bovis, M.leprae, M. avium, M. paratuberculosis, M. smegmatis); Legionella (e.g.L. pneumophila); Escherichia (e.g. enterotoxic E. coli, enterohemorragicE. coli, enteropathogenic E. coli); Vibrio (e.g. V. cholera); Shigella(e.g. S. sonnei, S. dysenteriae, S. flexnerii); Salmonella (e.g. S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis); Listeria (e.g. L.monocytogenes); Helicobacter (e.g. H. pylori); Pseudomonas (e.g. P.aeruginosa); Staphylococcus (e.g. S. aureus, S. epidermidis);Enterococcus (e.g. E. faecalis, E. faecium), Clostridium (e.g. C.tetani, C. botulinum, C. difficile); Bacillus (e.g. B. anthracis);Corynebacterium (e.g. C. diphtheriae), and Chlamydia (e.g. C.trachomatis, C. pneumoniae, C. psittaci). Representative examples ofparasite pathogens include Plasmodium (e.g. P. falciparum), Toxoplasma(e.g. T. gondii) Leshmania (e.g. L. major), Pneumocystis (e.g. P.carinii), Trichomonas (e.g. T. vaginalis), Schisostoma (e.g. S.mansoni). Representative examples of fungi include Candida (e.g. C.albicans) and Aspergillus.

Examples of autoimmune diseases include, but are not limited to,multiple sclerosis (MS), scleroderma, rheumatoid arthritis, autoimmunehepatitis, diabetes mellitus, ulcerative colitis, Myasthenia gravis,systemic lupus erythematosus, Graves' disease, idiopathicthrombocytopenia purpura, hemolytic anemia, multiplemyositis/dermatomyositis, Hashimoto's disease, autoimmune hypocytosis,Sjogren's syndrome, angitis syndrome and drug-induced autoimmunediseases (e.g., drug-induced lupus).

Moreover, as mentioned above, the fusion protein, nucleic acid molecule,vector, infectious particle, host cell and/or composition of the presentinvention can be used as an adjuvant to enhance the immune response ofan animal or human organism to a particular antigen. This particular useof the present invention may be made in combination with one or moretransgenes or transgene products as defined above, e.g. for purposes ofimmunotherapy. Preferably, the active agent (e.g. fusion protein,infectious particle or pharmaceutical composition of the invention) isadministered in combination with one or more transgenes or transgeneproducts. Accordingly, there is preferably also provided a compositioncomprising in combination a transgene product (e.g. a viral antigen or asuicide gene product) and a fusion protein as well as a compositioncomprising vector(s) or viral particles encoding a transgene product anda fusion protein. The transgene and the fusion-encoding nucleic acidsequences may be expressed from the same vector or from separate vectorswhich may have the same origin (e.g. adenoviral vectors) or a differentorigin (e.g. a MVA vector encoding the particular antigen and anadenoviral vector encoding the fusion protein). The fusion protein andthe transgene product (or their respective encoding vectors) can beintroduced into the host cell or organism either concomitantly orsequentially either via the mucosal and/or systemic route.

A preferred combination in the context of the present invention uses acomposition comprising or encoding (i) a fusion protein having an aminoacid sequence as shown in any of SEQ ID NO: 1-19, and (ii) an RPVantigen (particularly preferred in this context is a nononcogenic andmembrane-anchored early antigen of HPV-16). For example, a host organismcan be treated with a vector which expresses the fusion protein of theinvention and either with a nononcogenic and membrane-anchored HPV-16 E7variant or a vector which expresses it. Alternatively, a host organismcan be treated with the fusion protein of the invention and either witha nononcogenic and membrane-anchored HPV-16 E7 variant or a vector whichexpresses it. Preferably, the fusion protein of the invention is encodedby an adenoviral vector and the HPV antigen by a MVA vector. In thisregard, the adenoviral vector encoding the fusion protein of theinvention is initially administered in the host organism and the MVAvector encoding the immunogenic HPV antigen is subsequently administeredto the same host organism after a period varying from one day to twomonths. The fusion protein-encoding adenoviral vector is preferablyadministered by the mucosal route whereas the MVA vector is injected bysubcutaneous route. Compositions comprising a unique vector containingthe sequences encoding both the fusion protein and a nononcogenic andmembrane-anchored HPV-16 E7 variant can also be envisaged in thiscontext. Booster vaccinations with the particular antigen or a vectorexpressing it can also be performed from about 2 weeks to several yearsafter the original administration.

Another preferred combination in the context of the present inventionuses (i) a fusion protein having an amino acid sequence as shown in anyof SEQ ID NO: 1-19 or a vector encoding it and (ii) a vector encodingthe FCU-1 gene product (Cdase-UPRTase fusion which is described inWO99/54481). Preferably, the fusion protein of the invention is encodedby an adenoviral vector and the FCU-1 gene product by a MVA vector. Inthis regard, both vectors can be co-administered or administeredsequentially in a short time period into the host organism; e.g. byintratumor injection. The prodrug 5-FC is then given to the hostorganism within the day or week following the administration of bothvectors.

The present invention also provides a method for the treatment of ahuman or animal organism, comprising administering to said organism atherapeutically effective amount of the fusion protein, the vector, theinfectious viral particle, the host cell or the composition of theinvention. As used herein a “therapeutically effective amount” is a dosesufficient for the alleviation of one or more symptoms normallyassociated with the disease or condition desired to be treated. Whenprophylactic use is concerned, this term means a dose sufficient toprevent or to delay the establishment of a disease or condition.

The method of the present invention can be used for preventive purposesand for therapeutic applications relative to the diseases or conditionslisted above. It is to be understood that the present method can becarried out by any of a variety of approaches. For this purpose, thefusion protein, the vector, the infectious viral particle, the host cellor the composition of the invention can be administered directly in vivoby any conventional and physiologically acceptable administration route,such as those recited above, using specific delivery means adapted tothis administration route. It could be advantageous to proceed to theadministration of the active agent following an increase of permeabilityof a blood vessel. Such a permeability increase may be obtained byenhancing hydrostatic pressure (i.e. by obstructing outflow and/orinflow), osmotic pressure (i.e. with hypertonic solution) and/or byusing appropriate drugs (e.g. histamine; WO 98/58542).

Alternatively, one may employ eukaryotic host cells that have beenengineered ex vivo to contain the active agent according to theinvention. The transfected/infected cells are grown in vitro and thenreintroduced into the patient. The graft of encapsulated host cells isalso possible in the context of the present invention (Lynch et al,1992, Proc. Natl. Acad. Sci. USA 89, 1138-1142). Cells infected ex-vivocan be either autologous cells or heterologous cells, e.g. heterologouscells obtained from one or a plurality of subjects with a conditionsimilar to that which is to be treated. The cells can be of a singlecell type or of a mixture of cell types, e.g. they can comprise cells ofone or plural cell lines established from clinical tumour samples. Thecells for administration can preferably be inactivated, e.g. byirradiation, before administration. Among the cells that can usefully betreated in this way are for example malignant cells of human ornon-human organisms (see R Jurecic et al, ch 2, pp 7-30 in ‘Somatic GeneTherapy’ CRC Press 1995, ed. P. L. Chang).

The efficacy of the active agent or composition of the present inventionto enhance the immune response in an animal or human organism can betested in a variety of ways including, but not limited to, detection ofcellular immunity within the treated organism, determining lymphocyte ordendritic cell activity, detection of immunoglobulin levels, determiningthe activity of antigen presenting cells, determining dendritic celldevelopment or challenge of the treated organism with an appropriateinfectious or tumor-inducing agent to determine whether the treatedorganism is resistant to disease. In one embodiment, therapeuticcompositions can be tested in animal models such as mice. Suchtechniques are known to those skilled in the art.

As discussed above, the method of the present invention is particularlyintended for the treatment of cancers, to provide tumor inhibitiongrowth or tumor regression. For example, tumor inhibition may bedetermined by measuring the actual tumor size over a period of time.More specifically, a variety of radiologic imaging methods (e.g., singlephoton and positron emission computerized tomography; see generally,“Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.)Springer-Verlag, New York, 1986), may be utilized to estimate tumorsize. Such methods may also utilize a variety of imaging agents,including for example, conventional imaging agents (e.g., Gallium-67citrate), as well as specialized reagents for metabolite or receptorimaging, or immunologic imaging (e.g., radiolabeled monoclonal antibodydirected to specific tumor markers). In addition, non-radioactivemethods such as ultrasound (see, “Ultrasonic Differential Diagnosis ofTumors”, Kossoff and Fulcuda, (eds.), Igaku-Shoin, New York, 1984), mayalso be used to estimate the size of a tumor. Alternatively, inhibitionof tumor growth may be determined based upon a change in the presence ofa tumor marker. Examples include PSA for the detection of prostatecancer and CEA for the detection of colorectal and certain breastcancers. For yet other types of cancers such as leukemia, inhibition oftumor growth may be determined based upon a decreased number of leukemiccells in a representative blood cell count.

Further validation of the therapeutic efficacy of the active agent ofthe invention for treating cancer can be determined in a suitable animalmodel, e.g. using mice injected with a representative human cancer cellline. After solid tumors have developed to a sizeable diameter, the miceare injected intravenously or intratumorally with the active agent, andthen monitored for reduced tumor growth rate and increased survival (seeExample 4).

Prevention or treatment of a disease or a condition can be carried outusing the present method alone or, if desired, in conjunction withpresently or conventional therapeutic modalities (e.g. radiation,chemotherapy and/or surgery). The use of multiple therapeutic approachesprovides the patient with a broader based intervention. In oneembodiment, treatment with an active agent according to the inventioncan be preceded by surgical intervention. In another embodiment,radiotherapy (e.g. gamma radiation) is provided in combination with theactive agents according to the invention. Those skilled in the art canreadily formulate appropriate radiation therapy protocols and parameterswhich can be used in the method of the invention (see for example Perezand Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed.JB Lippincott Co; using appropriate adaptations and modifications aswill be readily apparent to those skilled in the field). Preferably, theactive agent of the invention is administered before exposing theindividual to a therapeutically effective amount of anti-cancerradiation. In still another embodiment, the method of the invention isassociated to chemotherapy. Chemotherapy include administration ofcytotoxic and/or cytostatic agents which can be provided in a singledose or, alternatively, in multiple doses that are administered overseveral hours, days and/or weeks. Chemotherapeutics are deliveredaccording to standard protocols using standard agents, dosages andregimens and their administration may proceed, be concommitant, orsubsequent to the administration of the active agent of the invention.Suitable chemotherapeutics include without limitation cisplatin,carboplatin, doxirubicin, bleomycin, vinblastine, danurubicin,tamoxiphen, taxol, 5-FU and methotrexate. In some embodiments,chemotherapy and radiation treatments are both employed before orfollowing the administration of the active agent of the invention.

When the method of the invention uses a vector, infectious particle,host cell or composition engineered to express a transgene encoding asuicide gene product, it can be advantageous to additionally administera pharmaceutically acceptable quantity of a prodrug which is specificfor the expressed suicide gene product. The two administrations can bemade simultaneously or consecutively, but preferably the prodrug isadministered after the active agent of the invention. By way ofillustration, it is possible to use a dose of prodrug from 50 to 500mg/kg/day, a dose of 200 mg/kg/day being preferred. The prodrug isadministered in accordance with standard practice. The oral route ispreferred. It is possible to administer a single dose of prodrug ordoses which are repeated for a time sufficiently long to enable thetoxic metabolite to be produced within the host organism or cell. Asmentioned above, the prodrug ganciclovir or acyclovir can be used incombination with the TK. HSV-1 gene product and 5-FC in combination withthe cytosine deaminase and/or uracil phosphotransferase gene product.

The present invention also relates to a method for enhancing an immuneresponse in an animal or human organism comprising introducing into saidorganism the fusion protein, the vector, the infectious particles, thehost cells or the composition of the invention, so as to enhance saidimmune response. The immune response can be a specific and/or anonspecific, humoral and/or cell-mediated response. Specifically, theimmune response is a T cell response, and more specifically a cytotoxicT cell response. Preferably, the method of the invention allows toenhance the number and/or the cytolytic activity of CTLs specific for aselected antigen. Introduction is preferably made subcutaneously,intradermally, intramuscularly, intranasally, intratumorally or in closeproximity of a tumor. In one preferred embodiment, the method of theinvention is directed to enhancing an antigen-specific immune responsein a host cell or organism, by using an active agent comprising, orexpressing a transgene product consisting of one or more specificantigens against which a specific immune response is desired (e.g. anHPV-16 E6 or E7 variant). In another embodiment, the method of theinvention is directed to enhancing an antigen-specific immune responsein a host cell or organism, by using an active agent comprising orexpressing a transgene consisting of one or more tumor-associated ortumor-specific antigens present on a tumor, in order to inhibit growthor to prevent re-growth of any tumors bearing said antigen.

The present invention also provides the use of the fusion protein, thevector, the infectious particles, the host cells or the composition ofthe invention, for the preparation of a drug intended for the purpose ofactivating maturation of dendritic cells in an animal or human organism,and thus enhancing a nonspecific immune response against tumor or viralantigens. In a preferred embodiment, this use is intended to theprevention or treatment of a disease that can be reversed by theactivation of maturation of dendritic cells. An enhancement of thematuration of dendritic cells can be evaluated as illustrated in Example2. In one preferred embodiment, the fusion protein for this use isIL-2/IL-18 (with a special preference for the illustrated IL-2/proIL-18and IL-2/proIL-18(K89A) fusions) or IL-7/IL-2.

The present invention also provides the use of the fusion protein, thevector, the infectious particles, the host cells or the composition ofthe invention, for the preparation of a drug intended for the purpose ofactivating NKT cells in an animal or human organism, and thus enhancinga nonspecific immune response against tumor or viral antigens. In apreferred embodiment, this use is intended to the prevention ortreatment of a disease such as cancer and infectious disease that can bereversed by the activation NKT cells. An enhancement of the activationof NKT cells can be evaluated as illustrated in Example 2. In onepreferred embodiment, the fusion protein for this use is IL-2/IL-18(with a special preference for the illustrated IL-2/proIL-18 andIL-2/proIL-18(K89A) fusions).

The present invention also provides the use of the fusion protein, thevector, the infectious particles, the host cells or the composition ofthe invention, for the preparation of a drug providing lowercytotoxicity upon administration in an animal or human organism ascompared to the cytotoxicity observed upon administration of theindividual X and/or Y entities. A limited cytotoxicity is especiallyadvantageous for treating cancers and infectious diseases such as thosecited above. It can be evaluated by measuring AICD activity or VLS(Vascular Leak Syndrome) as illustrated in Example 3. In one preferredembodiment, the fusion protein for this use is IL-2/IL-18 (with aspecial preference for the illustrated IL-2/proIL-18 andIL-2/proIL-18(K89A) fusions) or IL-7/IL-2.

The invention also provides antibodies that selectively bind to thefusion protein of the present invention or peptide fragments thereof. Asused herein, an antibody selectively binds a target peptide when itbinds the target peptide and does not significantly bind to unrelatedproteins. In certain cases, it would be understood that antibody bindingto the peptide is still selective despite some degree ofcross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art. The antibodies of the present inventioninclude polyclonal antibodies and monoclonal antibodies, as well asfragments of such antibodies, including, but not limited to, Fab orF(ab′).sub.2, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target polypeptide/peptide. Several such methods are described byHarlow (1989, Antibodies, Cold Spring Harbor Press). A preferred methodto produce antibodies of the present invention includes (a)administering to an animal an effective amount of a fusion protein ofthe present invention and/or a peptide fragment thereof, to produce theantibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingconventional techniques in the art. The full-length fusion protein or anantigenic peptide fragment can be used. Antibodies are preferablyprepared from regions or discrete fragments of the secreted proteins.Particularly important regions and fragments are those comprising uniquesequences of the fusion proteins of the invention, such as the onesoverlapping the fusion site between X and Y entities. An antigenicfragment will typically comprise at least 8 contiguous amino acidresidues. The antigenic fragment can comprise, however, at least 10, 12,14, 16 or more amino acid residues.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as reagents in assays to detect a fusionprotein of the present invention, (b) as reagents in assays to modulatecellular activity through a fusion protein of the present invention,and/or (c) as tools to recover a fusion protein of the present inventionfrom a mixture of proteins and other contaminants. The use of anantibody of the present invention as reagent can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase. Examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin. Examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.Examples of bioluminescent materials include luciferase, luciferin, andaequorin. Examples of suitable radioactive material include ¹²⁵I, ¹³¹I,³⁵S or ³H.

The antibodies can be used to isolate one of the fusion proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the recombinantly produced fusion protein from culturedcells. Also, such antibodies can be used to detect protein in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression. Further, such antibodies are usefulto detect the presence or to assess the expression of one of the fusionproteins of the present invention in cells, biological samples ortissues of an individual over the course of a treatment. Additionally,such antibodies can be used to identify individuals that requiremodified treatment modalities. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the functionof the fusion protein of the invention.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced in a different way from what is specifically describedherein.

All of the above cited disclosures of patents, publications and databaseentries are specifically incorporated herein by reference in theirentirety to the same extent as if each such individual patent,publication or entry were specifically and individually indicated to beincorporated by reference.

LEGENDS OF FIGURES

FIG. 1 illustrates the schematic construction steps for generating anadenoviral vector encoding a fusion protein.

FIG. 2 illustrates the in vitro evaluation of the functionality ofIL-2-containing fusion proteins by measurement of T cell costimulation.“Spleno” represents splenocytes, “ConA” represents splenocytes activatedwith Concanavalin A, “Anti-CD3” represents splenocytes activated with amurine CD3-specific antibody, and “½” and “ 1/10” represent thedilutions of the viral supernatants used in this assay. “Empty Ad”represents a negative control devoid of fusion-encoding sequences.

FIG. 3 illustrates the in vitro evaluation of the functionality of IL-7containing fusion proteins by measurement of the proliferation ofpro-B-2E8 lymphoblast cells. “rMu IL-7” represents recombinant murineIL-7 (1 to 500 ng/ml), “p” represents pure viral supernatants and “½”and “ 1/10” represent the dilutions of the viral supernatants used inthis assay.

FIG. 4 illustrates the in vitro evaluation of the functionality of IL-18containing fusion proteins by measurement of the induction of IFN-gsecretion by ConA pre-activated murine splenocytes (Concanavaline A 10μg/ml; 24 h). The production of IFN-g is evaluated by ELISAimmunoassays. “ 1/20” and “ 1/50” represent the dilutions of the viralsupernatants used in this assay. IL-18 here represents proIL-18.

FIG. 5 illustrates the in vitro activation of splenocytes. Analysis ofIFNg secretion induce on ConA-primed (10 μg/ml) or unprimed splenocytesby A549 supernatants containing 20 ng/ml of mproIL-18(K89A), thecombination of mIL-2+mproIL-18(K89A), mIL-2/matureIL-18(Ad-mIL-2/IL-18), mIL-2/matureIL-18(K89A) (Ad-mIL-2/IL-18*),mIL-2/proIL-18 and mIL-2/proIL-18(K89A). As negative control,supernatant of control virus-infected A549 cells were used. Theseresults are representative of three different experiments with similarresults. IL-18* represents IL-18(K89A).

FIG. 6 illustrates the in vivo analysis of fusion cytokine systemic andcell toxicity. Assessment of VLS syndrome induced by i.v. treatment ofmice with 2·10⁹ iu of an empty Ad (a), Ad-mIL-2 (b), Ad-mproIL-18(K89A)(c), the combination of Ad-mIL-2+Ad-mproIL-18(K89A) (b+c),Ad-mIL-2/matureIL-18(K89A) (d) and Ad-mIL-2/proIL-18(K89A) (e).

FIG. 7 illustrates the immunoadjuvant effect of Ad-mIL-2/proIL-18(K89A)in combination with MVA-E7. As before, * represents the mutation (K89A).

FIG. 8 illustrates the immunoadjuvant effect of Ad-mIL-2/proIL-18(K89A)in combination with MVA-FCU1. As before, * represents the mutation(K89A).

FIG. 9 illustrates the antitumor activity following three intratumoralinjections of Ad expressing IL-15-containing fusions in mice hearingB16F10 tumors. G1 represents treatment with an Ad vector expressing notransgene (Ad empty), G2 represents treatment with an Ad vectorexpressing the mature murine IL-15 cytokine equipped at its N-terminuswith the IL-2 signal peptide (Ad-mIL-15), G6 represents treatment withan Ad vector expressing the fusion between the mature murine IL-15cytokine equipped at its N-terminus with the IL-2 signal peptide and themurine IL-7 (Ad-mIL-15/mL-7). G7 represents treatment with an Ad vectorexpressing the fusion between the murine IL-21 cytokine and the maturemurine IL-15 (Ad-mIL-21/mL-15). G10 represents treatment with an Advector expressing the fusion between the mature murine IL-15 cytokineequipped at its N-terminus with the IL-2 signal peptide and the murineproIL-18 variant K89A (Ad-mIL-15/proIL-18*).

FIG. 10 represents the intratumoral injection of adenovirus encoding newimproved IL-15-versions in mice bearing B16F10 tumors. G1 representstreatment with an Ad vector expressing no transgene (Ad vide), G2represents treatment with an Ad vector expressing the mature murineIL-15 cytokine equipped at its N-terminus with the IL-2 signal peptide(Ad-mIL-15 or Ad-spi12-IL-15). G3 represents treatment with an Ad vectorexpressing the mature murine IL-15 cytokine equipped at its N-terminuswith the IgG kappa light chain signal peptide (Ad-spvKL-IL-15). G4represents treatment with an Ad vector expressing the mature murineIL-15 cytokine equipped with the endogenous long form signal peptide(Ad-spLSP-IL-15). G5 represents treatment with an Ad vector expressingthe mature murine IL-15 cytokine equipped with the endogenous short fowlsignal peptide and splice (Ad-spLSP-IL-15 splice).

The following examples serve to illustrate the present invention.

EXAMPLES

The constructs described below are prepared according to the generaltechniques of genetic engineering and of molecular cloning, as detailedin Sambrook et al. (2001, Molecular Cloning; A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y.) or according tothe manufacturer's recommendations when a commercial kit is used. Thecloning steps using bacterial plasmids are preferably carried out in theE. coli strain 5K (Hubacek and Glover, 1970, J. Mol. Biol. 50, 111-127)or in E. coli strain BJ5183 (Hanahan, 1983, J. Mol. Biol. 166, 557-580).The latter strain is preferably used for homologous recombination steps.The NM522 strain (Stratagene) is suitable for propagating the M13 phagevectors. The PCR amplification techniques are known to those skilled inthe art (see for example PCR. Protocols—A guide to methods andapplications, 1990; Ed Innis, Gelfand, Sninsky and White, Academic PressInc). With respect to the repair of restriction sites, the techniqueused consists in filling the overhanging 5′ ends using the largefragment of E. coli DNA polymerase I (Klenow). The Ad5 nucleotidesequences are those disclosed in the Genebank database, under thereference M73260 or AY339865.

Materials and Methods

Cloning and Construction of Multifunctional Cytokine cDNAs.

Splenocytes from C57B16 mice were harvested and stimulated during 3 dayswith a mixture of concanavalin A (10 μg/ml, SIGMA) and murine IL-2 (10IU/ml, R&D Systems) or LPS (10 μg/ml, SIGMA) and murine GM-CSF (50IU/ml, R&D Systems). mRNA from activated splenocytes were then extractedusing RNA Now (Ozyme). Murine IFN-g, IL-2, IL-7, IL-15, IL-18 and IL-21cDNAs were amplified by RT-PCR (Platinum Quantitative RT-PCR,Thermoscript™ one step system, Invitrogen) using specificoligonucleotides based on the sequence data available in specializeddata banks. The mutated fowls of murine IL-2 (D20I, N88R, N88G andQ126M) and the mutated form of murine IL-18 (K89A) were made usingQuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.,USA). Two forms of murine IL-18 cDNA have been used for the fusionmolecules, one encoding the precursor pro-IL-18 and one encoding themature murine IL-18 (devoid of the prosequence). The murine secretableIL-15 is described in Fehniger et al. (2001, J. Exp. Med. 193, 219-231)and Suzuki et al. (2001, J. Leuk. Biol. 69, 531-537) The followingoligonucleotides were used to clone and mutate the cytokine sequences:

Murine IL-2 (SEQ ID NO: 20) 5′: otg14157 cggaattccacagtgacctcaagtcc (SEQID NO: 21) 3′: otg14158 ggggtaccccttatgtgttgtaag Murine IL-2 (N88G) (SEQID NO: 22) 5′: otg15485 gagaatttcatcagcggtatcagagtaactgttg (SEQ ID NO:23) 3′: otg15486 caacagttactctgataccgctgatgaaattctc Murine IL-2 (N88R)(SEQ ID NO: 24) 5′: otg15487 gagaatttcatcagccgtatcagagtaactgttg (SEQ IDNO: 25) 3′: otg15488 caacagttactctgatacggctgatgaaattctc Murine IL-2(Q126M) (SEQ ID NO: 26) 5′: otg15489ggagatggatagccttctgtatgagcatcatctcaacaagccc (SEQ ID NO: 27) 3′: otg15490 gggcttgttgagatgatgctcatacagaaggctatccatctcc Murine IL-2 (D20I)(SEQ ID NO: 28) 5′: otg15536 gagcagctgttgatgatcctacaggag (SEQ ID NO: 29)3′: otg15537 ctcctgtaggatcatcaacagctgctc Murine IL-7 (SEQ ID NO: 30) 5′:otg14438 ccgctcgagcggatgttccatgtttcttttagata (SEQ ID NO: 31) 3′:otg14439 cggggtaccccgttatatactgcccttcaaaat Murine IL-18 (SEQ ID NO: 32)5′: otg14440 ccgctcgagcggatggctgccatgtcagaaga (SEQ ID NO: 33) 3′:otg14441 cggggtaccccgctaactttgatgtaagttagtgagagtgaac Murine IL-18 (K89A)(SEQ ID NO: 34) 5′: otg14457 ccagactgataatatacatgtacgcagacagtgaagtaagagg(SEQ ID NO: 35) 3′: otg14458 cctcttacttcactgtctgcgtacatgtatattatcagtctggMurine mature IL-18 (without pro-sequence) (SEQ ID NO: 36) 5′: otg14657ggtggaggcggttcaggcggaggtggctctaactttggccgacttcactg (SEQ ID NO: 37) 3′:otg14656 ctaactttgatgtaagttagtgagagtgaac Murine IL-21 (SEQ ID NO: 38)5′: otg14436 ccgctcgagcggatggagaggacccttgtctg (SEQ ID NO: 39) 3′:otg14437 cggggtaccccgctaggagagatgctgatgaatcatc Murine IL-15 (SEQ ID NO :40) 5′: otg15138 ccgctcgagcggatgtacagcatgcagctcgc (SEQ ID NO: 41) 3′:otg15139 cggggtaccccgctacttgtcatcgtcgtcc

As described in FIG. 1, once amplified by RT-PCR, the sequences encodingthe two cytokine moieties (X and Y) were cloned in frame by PCRtechniques with a flexible linker (G₄S)² or (G₄S)³ present between them(Gillies et al., 2002, Cancer Immunol. Immunother. 51, 449-460), usingthe following oligonucleotides:

*Murine IL-2/L/IL-18 (SEQ ID NO: 42) 5′: otg14442ccgctcgagcggatgtacagcatgcagctcga (SEQ ID NO: 43) 5′L: otg14444ggtggaggcggttcaggcggaggtggctctatggctgccatgtcagaaga (SEQ ID NO: 44) 3′L:otg14443 agagccacctccgcctgaaccgcctccaccttgagggcttgttgagatga (SEQ ID NO:33) 3′: otg14441 cggggtaccccgctaactttgatgtaagttagtgagagtgaac MurineIL-18/L/IL-2 (SEQ ID NO: 32) 5′: otg14440ccgctcgagcggatggctgccatgtcagaaga (SEQ ID NO: 45) 5′L: otg14446ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcg (SEQ ID NO: 46) 3′L:otg14445 agagccacctccgcctgaaccgcctccaccactttgatgtaagttagtga gagtgaacat(SEQ ID NO: 47) 3′: otg14447 cggggtaccccgttattgagggcttgttgag MurineIL-2/L/mature IL-18 (native or K89A) (SEQ ID NO: 48) 5′: otg15657ggtggaggcggttcaggcggaggtggctctaactttggccgacttcactg (SEQ ID NO: 49) 3′:otg15656 ctaactttgatgtaagttagtgagagtgaac *Murine IL-2/L/IL-7 (SEQ ID NO:42) 5′: otg14442 ccgctcgagcggatgtacagcatgcagctcga (SEQ ID NO: 50) 5′L:otg14449 ggtggaggcggttcaggcggaggtggctctatgttccatgtttcttttag (SEQ ID NO:44) 3′L: otg14443 agagccacctccgcctgaaccgcctccaccttgagggcttgttgagatga(SEQ ID NO: 31) 3′: otg14439 cggggtaccccgttatatactgcccttcaaaat MurineIL-7/L/IL-2 (SEQ ID NO: 30) 5′: otg14438ccgctcgagcggatgttccatgtttcttttagata (SEQ ID NO: 45) 5′L: otg14446ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcg (SEQ ID NO: 51) 3′L:otg14450 agagccacctccgcctgaaccgcctccacctatactgcccttcaaaatt (SEQ ID NO:47) 3′: otg14447 cggggtaccccgttattgagggcttgttgag *Murine IL-2/L/IL-21(SEQ ID NO: 42) 5′: otg14442 ccgctcgagcggatgtacagcatgcagctcga (SEQ IDNO: 52) 5′L: otg14448 ggtggaggcggttcaggcggaggtggctctatggagaggacccttgtctg(SEQ ID NO: 44) 3′L: otg14443agagccacctccgcctgaaccgcctccaccttgagggcttgttgagatga (SEQ ID NO: 39) 3′:otg14437 cggggtaccccgctaggagagatgctgatgaatcatc Murine IL-21/L/IL-2 (SEQID NO: 38) 5′: otg14436 ccgctcgagcggatggagaggacccttgtctg (SEQ ID NO: 45)5′L: otg14446 ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcg (SEQ IDNO: 53) 3′L: otg14451 agagccacctccgcctgaaccgcctccaccggagagatgctgatgaatcatc (SEQ ID NO: 47) 3′: otg14447 cggggtaccccgttattgagggcttgttgag *MurineIL-2/L/IFN-g (SEQ ID NO: 42) 5′: otg14442ccgctcgagcggatgtacagcatgcagctcga (SEQ ID NO: 54) 5′L: otg14636ggtggaggcggttcaggcggaggtggctctatgaacgctacacactgcat cttgg (SEQ ID NO: 44)3′L: otg14443 agagccacctccgcctgaaccgcctccaccttgagggcttgttgagatga (SEQ IDNO: 55) 3′: otg14637 cggggtaccccgtcagcagcgactccttttccg MurineIFN-g/L/IL-2 (SEQ ID NO: 56) 5′: otg14639ccgctcgagcggatgaacgctacacactgcatcttgg (SEQ ID NO: 45) 5′L: otg14446ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcg (SEQ ID NO: 57) 3′L:otg14641 agagccacctccgcctgaaccgcctccaccgcagcgactccttttccgc (SEQ ID NO:47) 3′: otg14447 cggggtaccccgttattgagggcttgttgag *Murine IL-2/L/IL-15(SEQ ID NO: 42) 5′: otg14442 ccgctcgagcggatgtacagcatgcagctcga (SEQ IDNO: 58) 5′L: otg15140 ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcgc(SEQ ID NO: 44) 3′L: otg14443agagccacctccgcctgaaccgcctccaccttgagggcttgttgagatga (SEQ ID NO: 41) 3′:otg15139 cggggtaccccgctacttgtcatcgtcgtcc Murine IL-15/L/IL-2 (SEQ ID NO:40) 5′: otg15138 ccgctcgagcggatgtacagcatgcagctcgc (SEQ ID NO: 45) 5′L:otg14446 ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcg (SEQ ID NO:59) 3′L: otg15141 agagccacctccgcctgaaccgcctccacccttgtcatcgtcgtccttg (SEQID NO: 47) 3′: otg14447 cggggtaccccgttattgagggcttgttgag *MurineIL-7/L/IL-15 (SEQ ID NO: 30) 5′: otg14438ccgctcgagcggatgttccatgtttcttttagata (SEQ ID NO: 58) 5′L: otg15140ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcgc (SEQ ID NO: 51) 3′L:otg14450 agagccacctccgcctgaaccgcctccacctatactgcccttcaaaatt (SEQ ID NO:41) 3′: otg15139 cggggtaccccgctacttgtcatcgtcgtcc Murine IL-15/IL-7 (SEQID NO: 40) 5′: otg15138 ccgctcgagcggatgtacagcatgcagctcgc (SEQ ID NO: 50)5′L: otg14449 ggtggaggcggttcaggcggaggtggctctatgttccatgtttcttttag (SEQ IDNO: 59) 3′L: otg15141 agagccacctccgcctgaaccgcctccacccttgtcatcgtcgtccttg(SEQ ID NO: 31) 3′: otg14439 cggggtaccccgttatatactgcccttcaaaaat *MurineIL-21/L/IL-15 (SEQ ID NO: 38) 5′: otg14436ccgctcgagcggatggagaggacccttgtctg (SEQ ID NO: 58) 5′L: otg15140ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcgc (SEQ ID NO: 53) 3′L:otg14451 agagccacctccgcctgaaccgcctccaccggagagatgctgatgaatca tc (SEQ IDNO: 41) 3′: otg15139 cggggtaccccgctacttgtcatcgtcgtcc MurineIL-15/L/IL-21 (SEQ ID NO: 40) 5′: otg15138ccgctcgagcggatgtacagcatgcagctcgc (SEQ ID NO: 52) 5′L: otg14448ggtggaggcggttcaggcggaggtggctctatggagaggacccttgtctg (SEQ ID NO: 59) 3′L:otg15141 agagccacctccgcctgaaccgcctccacccttgtcatcgtcgtccttg (SEQ ID NO:39) 3′: otg14437 cggggtaccccgctaggagagatgctgatgaatcatc *MurineIL-15/L/IL-18 (native or K89A) (SEQ ID NO: 40) 5′: otg15138ccgctcgagcggatgtacagcatgcagctcgc (SEQ ID NO: 43) 5′L: otg14444ggtggaggcggttcaggcggaggtggctctatggctgccatgtcagaaga (SEQ ID NO: 59) 3′L:otg15141 agagccacctccgcctgaaccgcctccacccttgtcatcgtcgtccttg (SEQ ID NO:33) 3′: otg14441 cggggtaccccgctaactttgatgtaagttagtgagagtgaac MurineIL-18 (native or K89A)/L/IL-15 (SEQ ID NO: 32) 5′: otg14440ccgctcgagcggatggctgccatgtcagaaga (SEQ ID NO: 58) 5′L: otg15140ggtggaggcggttcaggcggaggtggctctatgtacagcatgcagctcgc (SEQ ID NO: 46) 3′L:otg14445 agagccacctccgcctgaaccgcctccaccactttgatgtaagttagtga gagtgaacat(SEQ ID NO: 41) 3′: otg15139 cggggtaccccgctacttgtcatcgtcgtcc

In each case, both types of fusion proteins (X-Y and Y-X) wereconstructed and assayed for biological and therapeutic activities. Eachcytokine was also cloned individually in the same adenoviral backbone toserve as control.

Adenovirus Production and Titration

The sequence encoding each fusion protein was inserted in an adenoviralshuttle plasmid containing a CMV-driven expression cassette surroundedby adenoviral sequences (adenoviral nucleotides 1-458 and nucleotides3328-5788, respectively) to allow generation of the vector genome byhomologous recombination (Chartier et al., 1996, J. Virol. 70,4805-4810). In the resulting adenoviral vectors, E3 (nucleotides28592-30470) and Eli (nucleotides 459-3327) are deleted, and the E1region is replaced by the expression cassette containing, from 5′ to 3′,the CMV immediate-early enhancer/promoter, a chimeric humanbeta-globin/IgG intron, the sequence encoding the fusion protein and theSV40 late polyadenylation signal. The recombinant adenoviruses weregenerated by transfecting the PacI linearized viral genomes into the PERC6 complementation cell line (Fallaux et al., 1998, Human Gene Therapy9, 1909-1917). Virus propagation, purification and titration were madeas described previously (Erbs et al., 2000, Cancer Res 60, 3813-3822).

Cell Culture

In the examples which follow, use is made of the human pulmonarycarcinoma cell line A549 (ATCC; CCL-185), the 2E8 murine lymphoblast(ATCC; TIB-239) and the murine 2B4.11 T cell hybridoma (Delgado et al.,2001, J. Immunol. 166, 1028-1040). The culturing conditions areconventional in the art. For illustrative purposes, the cells are grownat 37° C. in DMEM (Gibco) supplemented with 10% Fetal Calf Serum andantibiotics. Cells are transfected according to standard techniquesknown to those skilled in the art.

P815 murine mastocytoma (DBA/2; FcR+, H2D^(d), MHCI+, ICAM1+, CD48+),and B16F10 (C57B1/6; H2D^(b), MHCI−, MHCII−, ICAM1−, CD48−) are murinemelanoma cancer cell lines obtained from the American Type CultureCollection (ATCC, TIB-64 and ATCC, CRL-6475 respectively). RenCa murinerenal carcinoma (BALB-C; H2D^(d), MHCI+, MHCII+, Fas+) and TC1 murinetumor cell line are described in Dybal et al. (1992, J. Urol. 148,1331-1337) and Lin et al. (1996, Cancer Res. 56, 21-26), respectively.All cell lines were tested negative for mycoplasma using Hoechst dye,cell culture and PCR.

Antibodies and Cytokines

Biotin-labelled anti-murine IL-2 and anti-murine IFN-g were purchasedfrom R&D Systems (UK). Biotin-labelled anti-murine IL-18 and anti-murineIL-7 were purchased from Peprotech Inc. (USA). Purified rabbitpolyclonal anti-mouse IL-15 was purchased from eBioscience (USA).Purified goat anti-murine IL-21 was purchased from R&D Systems (UK).Biotin labelled anti-goat IgG or anti-rabbit IgG were purchased fromAmersham Life Sciences (USA).

PerCP-CY5.5, FITC or Phycoerythrine-labeled rat anti-mouse CD4, CD8,CD3, CD25, CD31, CD69, MAC1, CD11c, H-2K^(b)/D^(b), Ia^(b), NK-1.1,NK-T/NK cell antigen or unconjugated rat anti-mouse CD4 and CD8 wereused as defined by the manufacturer (Pharmingen; San Diego, Calif.;USA). Unconjugated rabbit anti-human CD3 (which cross reacts with mouseCD3) or rabbit anti-rat IgG and peroxidase-labeled goat anti-rabbit wereused at concentrations suggested by DAKO (Germany).

Measurement of T cell apoptosis (AICD) was made using the Annexin V-FITCapoptosis detection kit (Pharmingen, San Diego, Calif., USA).

Recombinant murine IFN-g, IL-2, IL-7, IL-21 were purchased from R&DSystems (UK). Recombinant murine IL-15 and IL-18 were purchased fromPeprotech Inc. (USA). Concanavalin A was used at 1 μg/ml and purchasedfrom SIGMA.

Analysis of Multifunctional Cytokine Expression

RenCa or A549 cells were infected in suspension with adenoviral vectorsas previously described at MOI (multiplicity of infection) of 50 (30 minincubation of cells with virus dilutions in 100 μl of PBS supplementedwith 2% FCS, 1% cations) (Erbs et al., 2000, Cancer Res. 60, 3813-3822).Cells were then cultured in complete medium containing 5% FCS for 48 h.RNAs from infected A549 cells were analysed by Northern Blot using³²P-labelled mouse cytokine DNA specific probes.

Expression of individual cytokines constituting each of the fusionprotein was analysed by Western blot according to the ECL™ Westernblotting protocol provided by Amersham Life Sciences (UK). A549 cellswere infected at an MOI of 50. Seventy-two hours after infection,supernatants were collected and the cells were washed once with PBS anddisrupted in sample buffer (Novex, Invitrogen, France) by sonication.Supernatants and cell extracts were collected and then analysed byWestern Blot on 4-12% Nupage gel (Novex, Invitrogen, France) usingspecific anti-mouse cytokines and the ECL detection system (AmershamLife Sciences).

In Vitro Biological Activity of Multifunctional Cytokines

T or B cell proliferation assay. Mouse spleen cell or 2E8 lymphoblastcell proliferation was assessed by the uptake of [³H]thymidine aspreviously described (Gillis et al., 1978, J. Immunol. 120, 2027-2032;Ishihara et al., 1991, Dev. Immunol. 1, 149-161). For T cellproliferation, splenocytes were pre-activated by low doses (20 ng/ml) ofmurine CD3 specific antibody (145-2C11, Pharmingen, San Diego, USA) aspreviously described (Ting et al., 1988, J. 1 mmol. 141, 741-748).CD3-activated splenocytes were mixed with the fusion cytokines to betested as contained in infected A549 supernatants. As positive control,spleen or 2E8 cells (5×10⁴ cells/well) were stimulated in completemedium with either ConA (10 μg/ml), 100 ng/ml recombinant murine IL-2 orvarious concentrations of murine IL-7 (R&D Systems, UK). After 96 hours,the cells were pulsed with 1 μCi/well [³H]thymidine. Incorporation of[³H]thymidine into the DNA of proliferating T cells was measured byharvesting cellular DNA onto glass filter paper (PHD harvester,Cambridge Technology, USA) after 4 hours and by counting theradioactivity in a liquid scintillation counter (Beckman, Germany). Allmeasurements were made in triplicate.

IFN-g secretion assay. The relative bioactivity of murine IL-18 wasdetermined by the ability of Ad-fusion supernatants (obtained frominfected cells) to augment IFN-g production in vitro (Okamura et al.,1995, Nature, 378, 88-91; Oshikawa et al., 1999, Proc. Natl. Acad. Sci.USA, 96, 13351-13356). In brief, mouse splenocytes were cocultured withCon A (1.25 μg/ml) in 24-well plated for 48 hr. Ad-fusion supernatantswere added to cell suspensions of Con A-primed splenocytes in 96-wellplates for 24 hr. The supernatants were collected and assayed by ELISAto detect IFN-g production (Quantikine-R&D Systems, Minneapolis, Minn.)

CTL and NK/NKT cell cytotoxicity assays. Activities of fusion cytokineswere also assayed for CTL and NK cytotoxicity as previously described(Paul et al., 2000, Cancer Gene Ther. 7, 615-623). Mouse splenocyteswere cocultured with Ad-fusion supernatants obtained from A549 infectedcells during 7 days. The cytotoxic activities of primed splenocytes weremeasured on P815-CTL target or YAC-NK target as previously described(Shortman et al., 1986, J. Immunol., 137, 798-804) using EuDTPAcytotoxicity assay (Wallac Lab., Turku, Finland) (Blomberg et al., 1993,J. Immunol. Methods, 160, 27-34).

Immunostimulation in vitro. In order to analyse the in vitro effect ofmultifunctional fusion cytokines, bone marrow derived dendritic cells orsplenocytes were incubated with Ad-fusion supernatants for 3 to 7 days.Phenotypic markers of maturation and/or activation of dendritic cell,others APCS, B, T (CD4 and CD8), NK, and NKT cells were analysed usingmouse-specific antibodies by flow cytometry analysis (FACScan, BectonDickinson, USA).

ELISA Assay

Fusion cytokine concentrations were estimated by ELISA immunoassay.Briefly, dilution of the fusion containing supernatants were coated on amaxisorp 96 well plate (NUNC) overnight at 4° C. Fusion cytokines werethen revealed with purified polyclonal rabbit anti-mouse IL-2 or IL-18(Biovision CA). Rabbit IgG were then revealed with a specific monoclonalanti-rabbit IgG conjugated with HRPO (Jackson Laboratories). Wellscoated with serial dilutions of recombinant murine IL-2 or murine IL-18,in tissue culture medium, were used as positive control (R&D Systems,Minneapolis, Minn.) to generate standard curves for the estimations offusokine concentrations.

AICD (Activation Induced Cell Death) Assay

AICD, in which signals normally associated with lymphocyte stimulationinstead result in the demise of the cell, has been proposed as amechanism of the deletion of antigen-specific lymphocytes. T cells canbe sensitive or resistant to AICD, and IL-2 can regulate thesusceptibility of T cells to AICD (Brunner et al., 1996, Int. Immunol.,8, 1017-1026; Nguyen et al., 2001, Immunology, 103, 426-434). Murine Tcell hybridomas are well documented model systems for the study of AICD.Most T cell hybridomas die within hours after activation by presentationof anti-TCR or anti-CD3 antibodies follows by IL-2 treatment. AICD couldbe characterized by the de novo synthesis of Fas (CD95) and its ligand(FasL) (Brunner et al., 1996, Int. Immunol., 8, 1017-1026). To comparethe susceptibility of murine T cell hybridoma to AICD, 2B4.11 Thybridoma cells (Delgado et al., 2001, J. Immunol. 166, 1028-1040) werecultured in anti-CD3 coated 96 well plates (145-2C11 antibody; 4 μg/ml)during 18 hours in complete medium. Then, supernatants from A549infected cells with either Ad encoding multifunctional cytokines orcontrol supernatants (Ad encoding individual mIL-2, mIL-7, mIL-18,mIL-21 or empty adenovirus) were added for a 18 hours additional period.Recombinant murine IL-2 (R&D Systems, UK) was also used as positivecontrol (10-20 ng/ml). AICD has been measured by flow cytometry analysisusing a phycoerythrine-labeled mouse anti-mouse FasL specific antibody(Kay-10, Pharmingen, San Diego, USA) and an FITC-labelled Annexin VApoptosis Detection kit (Pharmingen, San Diego, USA).

AICD was also measured in vivo after subcutaneous injection ofadenoviruses encoding multifunctional fusion cytokines. In brief,C57BL/6 mice were injected one time subcutaneously with 2·10⁸ iu ofAd-fusion (or as a control Ad encoding individual mIL-2, mIL-7, mIL-21or empty adenovirus). Draining lymph nodes were then taken at differenttimes post-injection (5, 8 and 18 hours). AICD was measured as describedbelow on lymphocytes contained in the lymph node.

Quantification of VLS (Vascular Leak Syndrome Assay)

Vascular leak was studied by measuring the extravasation of Evans bluewhich, when given i.v., binds to plasma proteins, particularly albumin,and following extravasation can be detected in various organs asdescribed (Rafi-Janajreh et al., 1999, J. Immunol. 163, 1619-1627).Vascular leak was induced by injecting i.v. 2·10⁹ iu of murine IL-2encoding adenoviral vector once per day for three days. Groups of fiveC57B1/6 mice were injected i.v. with PBS, empty adenovirus, Ad-mIL-2,Ad-mIL-2+Ad-mproIL-18 or Ad-fusions. On day 4, mice were injected i.v.with 0.1 ml of 1% Evans blue in PBS. After 2 h the mice were bled todeath under anesthesia, and the heart was perfused with heparin in PBS.The lungs and liver, where maximum extravasation is known to occur, wereharvested and placed in formamide at 37° C. overnight. The Evan's bluein the organs was quantified by measuring the absorbance of thesupernatant at 650 nm. The VLS seen in Ad-cytokine treated mice wasexpressed as the percent increase in extravasation compared with that inPBS-treated controls. For histopathological studies, groups of fiveseparate mice were injected with empty Ad or PBS, Ad-mIL-2,Ad-mIL-2/mL-7, Ad-mIL-2/proIL-18(K89A) and Ad-mIL-2/matureIL-18(K89A) asdescribed earlier, and on day 4 lungs and liver were fixed in 10%formalin solution. The organs were embedded in paraffin, sectioned, andstained with hematoxylin and eosin. Perivascular infiltration was scaledby counting the number of lymphocytes infiltrating the vessel andaveraging the minimum and maximum range for each group. Sera frominjected mice were also collected for ASAT and ALAT measurement.

In Vivo Experiments

Murine P815, B16F10, RenCa and TC1 tumor cells were trypsinized, washed,and resuspended in PBS at 3×10⁶ cells/ml. One hundred microliter of thecell suspension was then injected subcutaneously into the right flank of6- to 7-week-old immunocompetent B6D2 mice. At day 7, 8 and 9 afterinjection, when tumors became palpable, the mice received threeintratumoral injections of 5×10⁸ iu of Ad-fusion or Ad controls dilutedin 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂. Tumors size and survival rate wereevaluated for a 120 day time period.

For evaluation of the immunoadjuvant effect of Ad-fusions in combinationwith MVA-E7 vector, one hundred microliter of the TC1 cell suspension(3×10⁶ cells/ml) was injected intravenously into the tail vein of 6- to7-week-old immunocompetent B6D2 mice. 39 days after injection, the micereceived three intranasal injections of 5×10⁸ iu of Ad fusion(Ad-mIL-2/proIL-18(K89A)) diluted in 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂at days 39, 46 and 53 and three subcutaneous injections of 5×10⁶ pfu ofMVA-E7 at days 42, 49 and 56. Tumors size and survival rate wereevaluated for a 120 day time period.

For evaluation of the immunoadjuvant effect of Ad-fusions in combinationwith MVA-FCU-1, one hundred microliter of the B16F10 cell suspension(3×10⁶ cells/ml) was injected subcutaneously into the right flank of 6-to 7-week-old immunocompetent B6D2 mice. At day 7, 8 and 9 afterinjection, when tumors became palpable, the mice received threeintratumoral injections of 5×10⁸ iu of Ad-fusion(Ad-mIL-2/proIL-18(K89A)) diluted in 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂and 10⁷ pfu of MVA-FCU1. The prodrug 5-FC was given in the feeding waterat a final concentration of 0.5%. Tumors size and survival rate wereevaluated for a 80 day time period.

The statistical difference in the in vivo survival experiments betweenthe different groups was assessed using Fischer exact application(Statistica 5.1 software, Statsoft Inc.) of the Kaplan-Meir survivalcurves. A P5.0.05 was considered statistically significant.

Histology, Immunohistochemistry or Flow Cytometry Analysis of In VivoResponse.

Tumors were established and injected with the various viruses asdescribed above for in vivo experiments. On day 13, tumors were measuredand excised. Tumor draining lymph nodes were also taken at the sametime. For flow cytometry analysis, tumors were disrupted by collagenase(SIGMA) digestion, cells were stained with the indicated antibodies andpopulation analysed by cytofluorimetry (Paul et al., 2002, CancerImmunol. Immunother. 51, 645-654).

Tumor P815 tissues were removed and directly embedded in OCT Compound onisopentane cooled on dried-ice. 5 μm sections were used forHematoxylin-Eosin staining (structural observations by light microscopy)or for immunohistochemistry. Infiltrating cells and blood vesselsdetection were performed on methanol-acetone (50:50) fixed cryosectionsusing following antibodies: rat anti-mouse CD4 (n^(o) 553727-Pharmingen)at a dilution of 1/500, rat anti-mouse CD8 (n^(o) 553027-Pharmingen) ata dilution of 1/500, rabbit anti-human CD3 (N1580-1/50 diluted-Dako)non-diluted, hamster anti-mouse CD11c (n^(o) 553799-Pharmingen) at adilution of 1/100, rat anti-mouse Ia-Ie (n^(o) 556999-Pharmingen) at adilution of 1/500, rat anti-mouse CD25-FITC (Pharmingen) at a dilutionof 1/50, goat anti-mouse IL18-R (AF856—R&D Systems) at a dilution of1/50, anti-mouse CD31 (n^(o) 01951D-Pharmingen) at a dilution of 1/50and rabbit anti-human von Willebrand factor (A0082-Dako) at a dilutionof 1/100. First antibodies were incubated for 1 h 30 at roomtemperature, rinsed in TBS-0.1% Tween20. The primary antibodies wererevealed by specific secondary antibodies rabbit anti-rat Ig(Z0494-Dako) at a dilution of 1/500, rabbit anti-hamster Ig (n^(o)6074102-Rockland) at a dilution of 1/500, horse anti-goat biotinylated0.5% (Vectastain Elite PK6200-Vector) or rabbit anti-FITC HRP(P0404-Dako) coupled at a dilution of 1/100, incubated for 30 minutesand then rinsed in buffer. Horseradish peroxidase (HRP)-labeled polymerconjugated with the second rabbit antibody (EnVision+System n^(o)K4003-Dako) or Streptavidin-HRP (Vector) was applied for 30 minutes,then rinsed and DiAminoBenzidine (DAB) was used as substrate. All slideswere counterstained with Hematoxylin.

Example 1 Construction of Adenoviruses Expressing Multifunctional FusionCytokines

The sequence encoding the multifunctional fusion cytokines wereconstructed as outlined in FIG. 1 and in Material and Methods. Thefusions generated are listed below: mIL-2/mIFN-g, mIFN-g/mL-2,mIL-2/mL-7, mIL-2/mL-21, mIL-21/mL-2, mIL-2/mL-15, mIL-15/mL-2,mIL-7/mIL-15, mIL-15/mIL-7, mIL-15/mL-21, mIL-21/mL-15, mIL-2/mproIL-18,mproIL-18/mL-2, mIL-2/m matureIL-18, m matureIL-18/mIL-2,mIL-2/mproIL-18(K89A), mproIL-18(K89A)/mIL-2, mIL-2/matureIL-18(K89A),matureIL-18(K89A)/mL-2. Fusion cytokines containing murine IL-2 mutants(D20I, N88R, N88G and Q126M) were also generated.

The sequence encoding each of these multifunctional cytokines was clonedin an adenovirus shuttle plasmid and used to generate E1 and E3-deletedadenovirus vectors. Single control cytokines were also cloned in anadenovirus shuttle plasmid (Ad-mIL-2, Ad-mIL-2 (D20I), Ad-mIL-2 (N88G),Ad-mIL-2 (N88R), Ad-mIL-2 (Q126M), Ad-mIFN-g, Ad-mIL-7, Ad-mIL-15,Ad-mIL-18, Ad-mIL-18 (K89A) and Ad-mIL-21).

Expression of multifunctional fusion cytokines in A549 cells infectedwith the different adenovirus vectors was analysed by Northern andWestern Blot. Northern Blot analysis revealed the correct size ofspecific mRNA of each fusion cytokine and of each control cytokine.Western Blot analysis using cytokine specific antibodies revealed amajor band having the expected molecular weight for each individualfusion. In some cases, additional bands were observed, reflectingalternative splicing events or different glycosylation pattern. Highexpression and secretion levels were detected for almost all fusions(higher levels are detected in supernatants of cells infected withAd-mIL-2/mproIL-18 and Ad-mIL-2/mproIL-18(K89A)), except for some of theIL-15 containing fusions and Ad-mIL-15 (see Example 6).

Amounts of secreted recombinant fusion cytokines were measured using aspecific ELISA assay of culture supernatants after infection of A549cells with adenoviral vectors encoding the IL-2/IL-18 fusion cytokinesand compared to the amount secreted by cells infected with Ad encodingthe individual cytokines.

TABLE 1 mIL-2 (ng/ml) mIL-18 (ng/ml Empty Ad 0  0 Ad-mIL-2 7 · 10⁴  0Ad-m mature IL-18 0 6 · 10⁴ Ad-mproIL-18 0 250 Ad-mproIL-18(K89A) 0 200Ad-mIL-2/matureIL-18 3 · 10³ 2.5 · 10³   Ad-mIL-2/matureIL-18 (K89A) 4 ·10³ 4 · 10³ Ad-mIL-2/m proIL-18 3 · 10⁴ 3 · 10⁴ Ad-mIL-2/m proIL-18(K89A) 2.5 · 10⁴   2.5 · 10⁴  

As was observed by Western Blot analysis, mIL-2/mproIL-18 andmIL-2/mproIL-18(K89A) were expressed in Ad-infected cells at highestlevels in approximately the same range as mIL-2 alone but 100 times morethan Ad-proIL-18 alone. Expression of mIL-2/matureIL-18 andmIL-2/matureIL-18(K89A) was approximately one tenth that obtained withAd-mIL-2 indicating that the lack of a prosequence is deleterious to theexpression at least in the adenovirus system.

Stability of recombinant fusion cytokines was also assessed in vitro byWestern Blot analysis. A549 cells were infected with Ad-mIL-2 orAd-mproIL-18(K89A) alone, or the combination ofAd-mIL-2+Ad-mproIL-18(K89A) or with an adenovirus expressing theIL-2/IL-18 fusion (Ad-mIL-2/mproIL-18(K89A)). Supernatants were analyzedafter 24 h, 48 h and 72 h post infection. Blots were probed with (a) arabbit anti-mouse IL-2 antibody or (b) a rabbit anti-mouse IL-18antibody Unexpectedly, a higher stability of IL-2 expression wasobserved for the mIL-2/mproIL-18(K89A) fusion protein as compared to thecytokine alone or the combination of the two cytokines. Moreover, ahigher IL-18 expression was also observed when the proIL-18(K89A) entityis expressed as a fusion with mIL-2 (Ad-mIL-2/proIL-18(K89A) construct)rather than when expressed individually (Ad-mproIL-18(K89A) construct).On the basis of this results, it seems that the fusion of IL-2 withIL-18 allows to maintain a fixed ratio of both mIL-2 and mproIL-18(K89A)in contrast to the combination of Ad-mIL-2+Ad-mproIL-18(K89A). Theexpression of the fusion cytokine mIL-2/mproIL-18(K89A) was alsoevaluated by RT-PCR. Immunocompetent B6D2 mice bearing palpable P815tumors were injected with 5×10⁸ iu of empty Ad, Ad-mIL-2,Ad-mIL-2+Ad-mproIL-18(K89A) or Ad-mIL-2/mproIL-18(K89A). Tumors wereremoved 72 hours after injection and mRNA were extracted. RT-PCR wascarried out using oligonucleotide probes specific for mIL-2, mproIL-18or sequences specific to the mIL-2/mproIL-18(K89A) fusion. As before,the injection of Ad-mIL-2/mproIL-18(K89A) resulted in the maintenance ofa fixed ratio of both mIL-2 and mproIL-18(K89A) in contrast to thecombination of Ad-mIL-2+Ad-mproIL-18(K89A).

Example 2 In Vitro Functionality of the Fusion Cytokines

In vitro functionality of IL-2-containing fusions

The effect of fusion proteins on T cell stimulation was analysed byassessing the proliferation of murine splenocytes when incubated withanti-CD3 plus Ad-fusion cytokine supernatants as described in Materialand Methods. IL-2 is known to be a strong inducer of CD3-pre-activatedsplenocyte proliferation. Briefly, the proliferation of murinesplenocytes incubated with Ad-fusion supernatants was measured in a Tcell proliferation assay. Supernatant concentrations were adjusted tohave equivalent (20 μg/ml) content of total cytokine or fusion cytokineAs illustrated in FIG. 2, a strong stimulation index was obtained withAd-mIL-7/IL-2, and Ad-mIL-2/mproIL-18 supernatants (2, and 1.8respectively), which was higher than that obtained with Ad-mIL-2. The Tcell proliferation activity of the Ad-fusions expressing the IL-18variant (K89A) was also analysed by comparison to Ad expressingindividual cytokines (Ad-mIL-2, Ad-mproIL-18(K89A)) and the combinationof the two (Ad-m-IL-2+Ad-mproIL-18(K89A)). The results confirm astronger stimulation index provided by Ad-mIL-2/mproIL-18(K89A) than forAd-mIL-2, Ad-mproIL-18(K89A) or the combination of the two. On the otherhand, supernatants containing the fusions IL-21/IL-2, IL-15/IL-7,IL-2/IL-15 and IL-15/IL-21 show stimulation indices comparable withthose obtained for IL-2 supernatants. No proliferation was observed withan empty virus supernatant.

In Vitro Functionality of IL-7-Containing Fusions

The in vitro functionality of IL-7-containing fusions was evaluatedusing an IL-7 dependent cell line—the murine pro-B 2E8 cell line—, whichis able to grow only in the presence of IL-7 in the medium. The abilityof supernatants of A549 cells infected with Ad-mIL-2/IL-7 andAd-mIL-7/IL-2 to promote 2E8 proliferation was tested and compared tothe Ad-mIL-7 supernatants and recombinant IL-7 as positive controls andan empty Ad as negative control.

As expected, recombinant murine IL-7 induced the proliferation of 2E8 asAd-mIL-7 supernatant. As illustrated in FIG. 3, the proliferation rateof 2E8 treated with Ad-IL-2/IL-7 supernatants is higher than thatobtained with Ad-mIL-7 supernatant at the same dilutions. As a result,the proliferation rate obtained with 1/10-diluted Ad-IL-2/IL-7,Ad-IL-7/IL-2 and Ad-IL-7 supernatants is comparable to that obtainedwith 20, 10 and 15 ng/ml of recombinant murine IL-7 respectively. Noproliferation was observed with an empty virus supernatant.

In Vitro Functionality of IL-18-Containing Fusions

IL-18 is described as a strong inducer of IFN-g secretion both in vitroand in vivo. To evaluate the biological activity of IL-18-containingfusions, secretion of murine IFN-g by conA-primed murine splenocytes wasquantified as described in Material and Methods. As a result and asillustrated in FIG. 4, 1/20-diluted supernatants containingAd-mproIL-18/IL-2 induced a higher concentration of murine IFN-g invitro (7 to 8 μg/ml/24 h/10⁶ cells) in comparison to those induced byAd-mIL-2 (4 μg/ml/24 h/10⁶ cells), Ad-mproIL-18 (2 μg/ml/24 h/10⁶ cells)and M-2/mproIL-18 (5.5 μg/ml/24 h/10⁶ cells). These differences arestatistically significant. The biological activity ofIL-18(K89A)-containing fusion cytokines was also assessed by evaluatingthe secretion of murine IFNγ by conA-primed splenocytes. As illustratedin FIG. 5, supernatants containing IL-2/IL-18 fusions (mIL-2/mproIL-18,mIL2/matureIL-18, mIL-2/mproIL-18(K89A) and mIL-2/matureIL-18(K89A),respectively) induce slightly higher levels of IFNg (approximately 100ng/ml/10⁶ cells) than supernatants containing mproIL-18(K89A) alone orthe combination of AdmIL-2+Ad-mproIL-18(K89A) (approximately 80 and 60ng/ml/10⁶ cells respectively).

In a second series of experiments, the secretion of IFNg was alsoquantified using unprimed murine splenocytes. Unexpectedly, asillustrated in FIG. 5, un-primed splenocytes were stimulated to secretehigh level of IFNγ only after activation with Ad supernatants containingmIL-2/mproIL-18, mIL-2/mproIL-18(K89A), mIL-2/matureIL-18 andmIL-2/matureIL-18(K89A) fusions cytokines. This suggests a novelactivity associated with the IL-2/IL-18 fusion cytokines, which is notseen with individual cytokines or a mixture of the two.

In Vitro Functionality of IFN-g-Containing Fusions

The functionality of the IFN-g gene product contained in the fusions ofthe invention was estimated using the ability of this cytokine toupregulate activation markers on APCs and tumor cells. In a simpleexperiment, Ad-fusion supernatants were added to murine splenocytes invitro during 72 hours, then the upregulation of activation markersspecific for murine splenocytes, APCs and CD8+ lymphocytes was assessedby flow cytometry analysis for change in T lymphocytes (CD8+), anddendritic cell (CD11b) as well as MHC class I, MHC class II markersusing specific antibodies as described in Material and Methods.

TABLE 2 Upregulation of activation markers on murine splenocytes Adfusion MHCI+ MHCII+ CD11b+ CD8+ IFN-g rec + + − − Empty Ad ++ ++ + +Ad-mIL-2 + + − ++ Ad-mIFN-g +++ ++ + + Ad-mIL-2/IFN-g ++++ ++++ +++ +++Ad-mIFN-g/IL-2 +++ +++ ++ +++ = no positive cells + = between 1 to 5% ofpositive cells ++ = between 10 to 20% of positive cells +++ = between 20to 40% of positive cells ++++ = more than 40% of positive cells

As illustrated in Table 2, supernatants of cells infected withAdIL-2/IFN-g fusion are most potent to induce the upregulation of MHCclass I and class II molecules in vitro but also unexpectedly toincrease dramatically the number of APCs (CD11b⁺) and CD8⁺ Tlymphocytes. The Ad-IFN-g/IL-2 supernatants induce the same level ofresponse as Ad-IFN-g with respect to these markers. IL-2 induces a lowlevel of activation of these cell populations.

In Vitro Ability of Fusion Proteins to Increase of Effector CellCytotoxicity

Activities of multifunctional cytokines were assayed for CTL and NKcytotoxicity as described in Material and Methods. Supernatants fromA549 cells infected with Ad-fusion were incubated during 7 days withmurine splenocytes. The results are summarized in Table 3.

TABLE 3 Increase of effector cell cytotoxicity Ad-fusion CTL activity NKactivity Empty Ad − − Ad-mIL-2 ++ +++ Ad-mIFN-g − + Ad-mIL-7 − −Ad-mproIL-18 ++ ++ Ad-mIL-21 − ++ Ad-mIL-2/IFN-g + ++ Ad-mIFN-g/IL-2 ++++ Ad-mIL-2/IL-7 + + Ad-mIL-7/IL-2 +++ ++++ Ad-mIL-2/proIL-18 +++ +++Ad-mproIL-18/IL-2 + ++ Ad-mIL-2/IL-21 ++ + Ad-mIL-21/IL-2 − ++ = nospecific lysis + = between 20 to 40% lysis to an E/T ratio of 50/1 ++ =between 40 to 60% lysis to an E/T ratio of 50/1 +++ = between 60 to 80%lysis to an E/T ratio of 50/1 ++++ = between 80 to 100% lysis to an E/Tratio of 50/1

As shown in Table 3, supernatants from A549 infected cells withAd-mIL-7/IL-2 and Ad-mproIL-18/IL-2 induced a high cytotoxic activityboth on CTL and NK activity in vitro. These activities are highlysuperior to those obtained with Ad-mIL-2, Ad-mIL-7 and Ad-mproIL-18supernatants. Moreover, the Ad-mIFN-g/IL-2 supernatants induced a highresponse on NK cytotoxicity but not on CTL response.

In addition, the effect of the mIL-2/mproIL-18(K89A) fusion was assessedfor CTL and NK cytotoxic activities and compared to that of eachcytokine alone (mIL-2 or mproIL-18(K89A) respectively), or thecombination of mIL-2+ mproIL-18(K89A). Murine splenocytes were culturedfor 7 days with supernatants from A549 cells infected with thecorresponding Ad (Ad-mIL-2, Ad-mproIL-18(K89A),Ad-mIL-2+Ad-mproIL-18(K89A) and Ad-mIL-2/mproIL-18(K89A)). Supernatantconcentrations were adjusted to have equivalent (20 μg/ml) content oftotal cytokine or fusokine. The results show that supernatants from A549infected cells with Ad-mIL-2/proIL-18(K89A) fusion induced cytotoxicactivity on both P815 and YAC target cells. Unexpectedly, the lyticactivity by splenocytes cultured with mIL2/proIL18(K89A) fusion wasgreater than that observed by splenocytes cultured with supernatantscontaining individual cytokines or the mixture mIL-2+mproIL-18(K89A).

Induction of CD8, NK and NKT Cells

The capacity of the fusion cytokines to induce proliferation of bothinnate and adaptative immune effector cells was evaluated. For thispurpose, the percentage of CD8 T lymphocytes, NK and NK/NKT effectorcells was quantified by flow cytometry using murine splenocytes culturedfor one week with Ad-fusion supernatants. The results of this assay arepresented in Table 4

TABLE 4 Induction of CD8, NK and NKT proliferation Ad-fusion CD8 (%) NK(%) NK-T/NK (%) mIL-21 rec 27 5 25 Empty Ad 14 3 7 Ad-mIL-2 58 5 11Ad-mproIL-18 41 16 45 Ad-mIL-21 49 13 38 Ad-mIL-2/mproIL-18 51 15 60Ad-mproIL-18/IL-2 55 14 7 Ad-mIL-2/IL-21 43 15 53 Ad-mIL-21/IL-2 45 1154 mIL-21 rec = recombinant murine IL-21 (20 ng/ml)

As illustrated in Table 4, all the Ad-fusion supernatants tested inducethe same proportion (approximately 50%) of CD8⁺ T lymphocytes (specificeffector cells) as Ad-mIL-2 supernatant. Moreover and in contrast toAd-mIL-2 or Ad-mIL-21, the Ad-fusion (Ad-mIL-2/mproIL-18, Ad-mIL-2/IL-21and Ad-mIL-21/IL-2) supernatants induce a very impressive proportion(>50%) of NK/NKT⁺ cells. NK1.1⁺ cells were also significantly induced inthe presence of Ad supernatants encoding these fusion proteins.

Moreover, it has been observed that incubation of murine splenocyteswith mIL-2/proIL-18(K89A) induces a dramatic increase in the percentageof both CD8⁺ (50%), NK⁺ (18%) and NK/NKT⁺ (51%) cells in comparison withsplenocytes cultured with empty Ad, Ad-mIL-2 andAd-mproIL-18(K89A)-generated supernatants.

Effect of the Fusion Cytokines on the Maturation of Murine DendriticCells

Bone marrow derived dendritic cells were obtained from C57B16 mice aspreviously described (Fields et al., 1998, J. Immunother. 21, 323-339).Immature dendritic cells were incubated with Ad-fusion supernatants for48 hours before phenotyping analysis by flow cytometry analysis.Upregulation of maturation factor of murine dendritic cells wasdetermined by measuring the percentage of CD80, CD86 and MHC II-Iabmarkers using specific monoclonal antibody (Pharmingen). Supernatantsobtained from cells infected with Ad-mIL-7/IL-2 andAd-mIL-2/mproIL-18(K89A) were shown to upregulate the CD80, CD86 andMHCII markers, reflecting maturation of murine DCs, although at aslightly lower level than a positive control (LPS, 1 μg/ml, DIFCO) orsupernatant from Ad-mIL-7.

In conclusion, adenovirus vectors expressing multifunctional cytokinesare fully functional, exhibit in some cases a higher biologicallyactivity than simply the additive activity of the individual cytokinesforming the fusion. Unexpected activities were also detected for some ofthese fusions, such as the ability of the IL2/IL-18 fusion (especiallymIL-2/mproIL-18(K89A)) to activate murine NKT cells and the ability ofIL-7/IL-2 and IL2/IL-18 (especially mIL-2/mproIL18 andmIL-2/mproIL-18(K89A)) fusions to induce murine DC maturation.

Example 3 Toxicity of Fusion Cytokines

In addition to its role in the initial activation of T and NK cells,IL-2 has a critical role in the maintenance of peripheral tolerance(Lenardo, 1996, J. Exp. Med. 183, 721-724). In this respect, IL-2 has acentral importance in Fas-mediated activation-induced cell death (AICD),a process that leads to the elimination of self-reactive T cells(Lenardo, 1996, J. Exp. Med. 183, 721-724; Van Parijs et al., 1999,Immunity 11, 281-288). As a result of this pivotal role in AICD, the Tcells generated in response to tumor vaccines containing IL-2 mayinterpret the tumor cells as self and the tumor-reactive T cells may bekilled by AICD-induced apoptosis.

It has been described recently that AICD limits effector function of CD4tumor-specific T cells and decrease T cell effector activity (Saff etal., 2004, J. Immunol. 172, 6598-6606). IL-2 is also known to becritically required for the activation of CD4+ CD25+ T cell suppressorfunction (Thorton et al., 2004, J. Immunol. 172, 6519-6523; Shimizu etal., 1999, J. Immunol. 163, 5211-5218). Although IL-2 therapy hasyielded encouraging results in the treatment of certain types of cancer,its use is limited by dose-dependent toxicity characterized by weightgain, dyspnea, ascites, and pulmonary edema. These signs of toxicityresult from increased capillary leak, also known as vascular leaksyndrome (VLS) (Rosenstein et al., 1986, J. Immunol. 137, 1735-1742;Baluna and Vitetta, 1997, Immunopharmacology 37, 117-132; Baluna et al.,1999, Proc. Natl. Acad. Sci. USA 96, 3957-3962). For this reason, thetoxicity of the fusion cytokines of the invention was compared to thatprovided by IL-2 in AICD and VLS assays.

AICD Assays

The percentage of two apoptotic markers (Annexin and Fas ligand (FasL))was evaluated in AICD assays both in vitro and in vivo, as described inMaterial and Methods. The results are presented in Table 5.

TABLE 5 In vitro toxicity of fusion cytokines. Measurement of AICD(results are presented as percentage of total gated cells) Ad-fusionAnnexin V+ FasL+ Medium 40 7 mIL-2 rec (10 ng/ml) 65 24 Empty Ad 42 8Ad-mIL-2 67 25 Ad-mIL-7 50 22 AdmIL-18 55 23 Ad-mIL-2/IL-18 48 18Ad-mIL7/IL-2 36 9

In vitro, Ad-mIL-7/IL-2 and Ad-mIL-2/IL-18 supernatants protect 2B4.11cells from AICD as reflected by the low level of the two apoptoticmarkers Annexin V and FasL (36 and 48% of Annexin V+ cells and 9 and 18%FasL+ cells, respectively). In marked contrast, treatment withrecombinant murine IL-2 and Ad-mIL-2 induced high apoptosis (65 and 67%of Annexin V+ cells and 24 and 25% FasL+ cells, respectively).

AICD was evaluated in vivo in the draining lymph nodes, 8 hours aftersubcutaneous injection of Ad-fusions or Ad-IL-2. Table 6 summarizes theresults obtained. The results are representative of two experiments,each with three mice.

TABLE 6 In vivo toxicity of fusion cytokines. Measurement of AICD(results are presented as percentage of total gated cells) Ad-fusionAnnexin V+ FasL+ Empty Ad 2 nt Ad-mIL-2 48 29 Ad-mIL-2/mproIL-18 19 18Ad-mIL-2/mproIL-18(K89A) 9 15 Ad-mIL7/IL-2 6 12

As illustrated in Table 6, flow cytometry analysis of the cellscontained in the lymph nodes revealed that injection of Ad-mIL-2 inducesa strong AICD in vivo (48% Annexin V⁺ and 29% FasL⁺ cells). In markedcontrast, IL-2/mproIL-18 (19% Annexin V⁺ and 18% FasL⁺ cells),mIL-2/mproIL-18(K89A) (9% Annexin V⁺ and 15% FasL+ cells) and evenbetter IL-7/IL-2 (6% Annexin V⁺ and 12% FasL⁺ cells) protect T cellsfrom IL-2 induced AICD.

In conclusion, both in vitro and in vivo AICD assays demonstrate the lowapoptosis status conferred by the fusion proteins of the invention.

VLS Assays

To assess the toxicity of candidate fusion cytokines, groups of healthyC57B1/6 mice were administered intravenously with high doses of empty Ador adenoviral vectors encoding mIL-2, mproIL-18(K89A), the combinationof mIL-2+ mproIL-18(K89A) and the fusion cytokines mIL-2/mIL7,mIL-2/mproIL-18(K89A) and mIL-2/matureIL-18(K89A). As illustrated inFIG. 6, the two fusion versions mIL-2/mproIL-18(K89A) andmIL-2/matureIL-18(K89A) induce much less vascular leak than does mIL-2and the combination of mIL-2+mproIL-18(K89A). A reduced vascular leakwas also observed in mice injected with Ad-mIL-2/mIL-7. These datademonstrate that the genetic fusions of IL-2 and proIL-18(K89A) as wellas IL-2 and IL-7 dramatically reduce cytokine toxicity associated withvasopermeability.

Moreover, the reduced toxicity provided by the fusion cytokine wasconfirmed by quantification of hepatic enzymes ASAT and ALAT in injectedmice sera. The results demonstrate the absence of hepatic toxicity aftertreatment with Ad-mIL-2 or Ad-Fusion cytokines.

Example 4 In Vivo Functionality of Fusion Cytokines

The anti-tumoral activity of the fusion cytokines of the invention wasinvestigated in four tumor models (P815, RenCa, B16F10 and TC1). Tumorswere established in B6D2 mice and tumor growth and mouse survival wereevaluated following three intratumoral injections of Ad-fusions (5×10⁸iu) for a 120 day time period. Table 7 summarizes the results obtainedin the four tumor models.

TABLE 7 Anti-tumor activity in murine tumor models (results areexpressed in percentage of tumor-free mice over a period of 120 days)Ad-fusion P815 B16F10 RenCa TC1 Ad-mIL-2 0 60 80 30 Ad-mIL-7 0 0 10  0Ad-mproIL-18 0 0 20 10 Ad-mproIL-18(K89A) 0 0 10 10 Ad-mIL-21 10 0 30 ntAd-mIFN-g 5 0 15 nt Ad-mIL-2 + Ad-mproIL-18 10 40 80 ntAd-mIL-2/mproIL-18 40 30 70 40 Ad-mIL-2/mproIL-18(K89A) 70 50 90 60Ad-mIL-7/IL-2 20 20 70 30 Ad-mIL-21/IL-2 nt 10 50 nt Ad-mIFN-g/IL-2 nt10 60 nt nt = not tested

As illustrated in Table 7, Ad-mIL-2/mproIL-18 is the most effectivefusion for curing tumors from various origins (especially murinemastocytomas (P815), renal carcinomas (RenCa) and HPV-transformed tumors(TC1). More importantly, the antitumoral protection observed for thisfusion cytokine is significantly higher than that conferred byadministration of a vector encoding the individual cytokines (seeAd-mIL-2 or Ad-mproIL-18) as well as the co-administration of vectorsencoding separately these cytokines (Ad-mIL-2+Ad-mproIL-18), at least inthe RenCa, P815 and TC1 tumor models. Moreover, the use of a mutatedform of IL-18 (K89A) dramatically increases the anti-tumor efficacy inall tumor model (see Ad-mIL-2/mproIL-18(K89A) providing 70% of tumorfree mice in P815 model, 50% in B16F10 model, 90% in the RenCa model and60% in the TC1 model). Significant anti-tumor activity was also observedin several animal models treated with Ad-mIL-7/IL-2. Ad-mIL-21/IL-2 andAd-mIFN-g/IL-2 also provide anti-tumor protection to the same extent asAd-mIL-2.

The in vivo depletion of CD8, NK and CD4 cells was performed asdescribed by Slos et al., 2001, Cancer Gene Ther. 8, 321-332). Thesurvival data show that the anti-tumor activity ofAd-mIL-2/mproIL-18(K89A) was strictly dependent on CD8 and NK cellactivity. Interestingly, CD4 depletion increased the in vivo activity ofintra-tumor administration of the fusion cytokine.

Importantly, it should be noted that no immune response against thefusion cytokine was observed in vivo in the serum of treated mice (datanot shown).

The in vivo anti-tumoral efficacy of the fusion cytokines was alsocorrelated with the analysis of intratumoral infiltrates and of proximalactivation of both innate and adaptative immune effector cells (in thedraining lymph nodes) by histology, immunohistochemistry or flowcytometry in the P815 model as described in Material and Methods. Theresults are presented in Table 8.

TABLE 8 Analysis of tumor infiltrates after intratumoral injection ofAd-fusions. Ad- Ad-mIL- Ad-mIL- Ad-mIL- NI empty Ad-mIL-2 7/IL-2 2/IL-182/IL-18* CD3 + + ++ ++ ++++ ++++ CD4 + + +++ +++ + ++ CD8 − − − + ++++++ CD25 + + + ++ +++ ++ Ia-Ie − + + + ++ +++ IL-18R + + + + ++++ +++CD-31 +++ +++ +++ +++ +++ ++ vonW + + + ++ +++ ++++ necrosis <5% <10%<10% <5% 30-40% 70-80% − = no positive cells + = between 1 to 5% ofpositive cells ++ = between 10 to 20% of positive cells +++ = between 20to 40% of positive cells ++++ = more than 40% of positive cells

As illustrated in Table 8, following Ad-fusion injections,immunohistochemistry analysis reveals that tumors injected withAd-mIL-2/mproIL-18 and Ad-mIL-2/mproIL-18 (K89A) are highly necrotic.Moreover, immuno-histology demonstrates pronounced changes in infiltratepatterns differing from initial tumor histology, with an increase in thenumbers of CD8⁺/CD25⁺-activated T cells, CD4⁺ T cells and APCs. Inaddition, injected tumors clearly show upregulation of the IL-18receptor. Such changes are also observed in P815 tumors injected withAd-mIL-2 and Ad-mIL-7/IL-2 although at a lower level than withAd-mIL-2/mproIL-18 or Ad-mIL-2/mproIL-18 (K89A). Surprisingly, tumorsinjected with Ad-mIL-2/mproIL-18(K89A) are highly positive for the vonWillebrand factor suggesting the formation of new blood vessels.

Similar results were observed in the P815 tumor draining lymph nodes.Further, intratumoral injections of Ad-mIL-2/mproIL-18 andAd-mIL-2/mproIL-18(K89A) do not induce any AICD in the tumor draininglymph nodes. This is in contrast with P815 tumors treated with Ad-mIL-2.Moreover, in mice treated with Ad-mIL-2/mproIL-18(K89A) andAd-mIL-7/IL-2, an increase of immune cells (×30 to ×40) was observed inthe lymph nodes, whereas a lower number of immune cells was detected inthe lymph nodes of mice treated with Ad-MIL-2. This augmentationcorrelates with a dramatic decrease of the number of tumor cells. Thisshows clearly the inverse correlation between the total number of cellsin the tumor and the total number of cells in the draining lymph node.The immune effector cells present in the lymph nodes followingintratumoral injection of Ad-mIL-2/mproIL-18 (K89A) and Ad-mIL-7/IL-2are mainly activated CD8+ T lymphocytes (CD3⁺/CD69⁺; CD8⁺/CD25⁺) andalso activated APCs such as mature dendritic cells (CD11c⁺/MHCII⁺). Theproportion and the number of these effector cells is higher followinginjection with Ad-mIL-2/mproIL-18(K89A) and Ad-mIL-7/IL-2 than withAd-mIL-2, Ad-mproIL-18(K89A), Ad-mIL-7 alone or the combination ofAdmIL-2+Ad-mproIL-18(K89A).

Example 5 Evaluation of the Immunoadjuvant Effect of Ad-Fusions forSpecific Immunotherapy

The immunoadjuvant effect of Ad-fusion was evaluated in the TC1metastatic model. TC1 cells were injected by the intravenous route inorder to establish metastasis in the lung of C57B16 mice. SeveralAd-fusion were administered 10 days later by the intranasal orintratracheal routes to allow the expression of fusion protein in themetastasis environment in the lungs and also to induce a mucosalimmunity. Administration of Ad-mIL-2/IL-18 by the intranasal or theintratracheal routes induced total IgA in the vaginal washes of thetreated mice 15 days after adenovirus administration. The level of totalIgA was similar following intranasal or intratracheal administration.Moreover, the rate of anti-adenovirus neutralizing antibody issignificantly lower after Ad-mIL-2/IL-18 administration than after emptyadenovirus or Ad-mIL-2 administration. These results could be ofimportance, in that they indicate that potentially re-administration ofthe fusion-encoding adenoviral vectors could be facilitated due to thelower humoral immune response against these vectors.

Moreover, RT-PCR analysis showed that these two “mucosal” routes allow avery good expression of the fusion cytokine IL-2/IL-18 in the lung andmore precisely in the TC1 metastasis present in the lung. Importantly,the expression of the fusion correlated a strong in vivo effect sincethe growth of TC1 metastasis was stopped in treated mice.

All together, these results indicate the potential utility of the fusioncytokines as adjuvant for cancer or viral vaccine.

The use of the fusion cytokine mIL-2/proIL-18(K89A) as a geneticadjuvant for a cancer-specific vaccine was also assessed. First,evaluation of the immunoadjuvant effect of Ad-fusions was performed inthe TC1 metastatic model in combination with a MVA vector expressing anon oncogenic and membrane anchored E7 antigen of HPV-16 strain drivenby the 7.5K promoter (see WO99/03885). The mice received threeintranasal injections of 5×10⁸ iu of Ad-mIL-2/proIL-18(K89A) at days 39,46 and 53 and three subcutaneous injections of MVA-E7 at days 42, 49 and56. Tumors size and survival rate were evaluated for a 120 day timeperiod. As illustrated in FIG. 7, the combination of a tumor specificantigen expressing vector as MVA-E7 with an adenovirus expressing aIL-2/IL-18 fusion strongly enhanced the tumor-specific immune responsein the highly late metastatic TC1 model. This combination could increasethe survival of treated animals and decrease the number of residualmetastasis.

The immunoadjuvant effect of Ad-mIL-2/proIL-18(K89A) was also evaluatedin in the B16F10 model in combination with a MVA vector expressing theFCU-1 suicide gene placed under control of the chimeric 11k/7.5Kpromoter (WO99/54481). One hundred microliter of the B16F10 cellsuspension (3×10⁶ cells/ml) was injected subcutaneously into the rightflank of 6- to 7-week-old immunocompetent B6D2 mice. At day 7, 8 and 9after injection, when tumors became palpable, the mice received threeintratumoral injections of 5×10⁸ iu of Ad-mIL-2/proIL-18(K89A) dilutedin 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂ and 10⁷ pfu of MVA-FCU1. Theprodrug 5-FC was given in the feeding water at a final concentration of0.5%. Tumors size and survival rate were evaluated for a 80 day timeperiod. As illustrated in FIG. 8, the combination ofAd-mIL-2/proIL-18(K89A) with MVA-FCU1 clearly improved the antitumoralefficacy of a suicide gene therapy approach. This is an indication ofthe potent adjuvant effect of the fusion cytokine mIL-2/proIL-18(K89A)with a chemotherapy-based strategy

These results clearly demonstrate the potential of the Ad-fusionmIL-2/proIL-18(K89A) of the present invention as a genetic adjuvant forvaccination in combination with immunogen (e.g. tumor-specific antigenssuch as HPV-16 E7) and for cancer-specific vaccination in combinationwith a suicide gene therapy and appropriate chemotherapy.

Example 6 IL-15 Containing Fusion Cytokines

The IL-15 containing Ad constructs are described in Example 1(mIL-2/mL-15, mIL-15/mL-2, mIL-7/mL-15, mIL-15/mL-7, mIL-15/mL-21,mIL-21/mL-15). In addition, the fusion of mIL-15 to mproIL-18(K89A) wasalso performed using the same construction schema described in Materialand Methods and in Example 1. It should be noted that in the constructswhere IL-15 is located at the NH2 terminus of the fusion (mIL-15/mIL-2,mIL-15/mIL-7, mIL-15/mproIL-18(K89A) and mIL-15/mIL-21), the IL-15entity is designed so as to comprise the IL-2 peptide signal that isfused in frame to the mature murine IL-15 (as depicted in SEQ ID NO_(S)5). The control Ad-mIL-15 also comprises the mature IL-15 preceded bythe peptide signal of IL-2.

Expression of the IL-15 containing fusions was determined by Westernblot in A549 cells infected with the different Ad-vectors as describedin Material and Methods. Low expression and secretion levels weredetected for most of the IL-15 containing fusions as well as Ad-mIL-15,except for mIL-15/mIL-7, mIL-21/mIL-15 and mIL-15/mproIL-18(K89A)fusions which were secreted at high levels into the culture medium ofinfected A549 cells.

The anti-tumoral activity of adenovirus encoding IL-15-based fusioncytokines was investigated in mice bearing B16F10 tumors that weretreated by three intratumoral injections. Tumor growth was evaluated for43 days post implantation. As illustrated in FIG. 9, intratumoralinjection of Ad-mIL-15/mL-7, Ad-mIL-21/mL-15 andAd-mIL-15/mproIL-18(K89A) allows a statistical control of tumor growthin the treated animals as compared to intratumoral injection of an Adwithout transgene (Ad-empty) or the control Ad-mIL-15 expressing mIL-15alone.

In order to improve secretion of IL-15 in the fusion, additionalconstructs were designed to evaluate other signal peptides. In theAd-mIL-15 construct, the IL-2 signal peptide was replaced by either theendogenous IL-15 peptide signal in its long version without (spLSP) orshort natural form (spLSP splice) (Kurys et al., 2000, J. Biol. Chem.275, 30653) or by a heterologous signal peptide obtained from Kappalight chain of a mouse immunoglobin G (spVKL) (Meazza et al., 2000, Int.J. Cancer 87, 574). The expression and secretion of IL-15 driven by therespective signal peptides was evaluated by Western blot and compared tothe original construct equipped with the IL-2 signal peptide. Theresults show that the use of the IL-15 endogenous peptide signal (shortversion) and especially the IgG signal peptide could improve importantlythe level of IL-15 secretion by a factor 3 to ten. Moreover, theanti-tumor activity of the adenovirus encoding IL-15 cytokine controlledby the various signal peptides was investigated in mice bearing B16F10tumors. As illustrated in FIG. 10, intratumoral injection the highlysecretable Ad-IL-15 construct using the IgG signal peptide vKL providesa much higher survival rate than the other forms of IL-15. Constructionof fusion cytokines incorporating the signal peptide vKL-IL-15 versionis being performed in order to improve the activity and immunoadjuvanteffect of IL-15/IL-7, IL-21/IL-15, and IL-15/proIL-18(K89A) fusions.

General Discussion

The availability of recombinant cytokines has enabled research intocytokine biology as well as their application in a clinical setting. Oneaspect which is becoming clear is that the systemic injection of largedoses of cytokines is associated with considerable toxicity, usually dueto or accompanied by, vascular leak syndrome. In addition to itssystemic toxicity, the therapeutic value of IL-2 is also limited by itsshort half life. One approach reported in the literature to overcome thetoxicity and short life problems is to fuse IL-2 to an antibody (IL-2immunocytokine) or a protein with a long half life, to target the fusionto a unique antigen/receptor within the body. In a different approach,the present invention provides a series of cytokine fusion proteins inan effort to combine cytokines which stimulate the innate immune systemwith cytokines which promote an adaptive immune response. A variety ofcytokines, including IFNg, IL-7, IL-12, IL-15, IL-18 and IL-21 weregenetically fused to IL-2 and produced using E1 and E3-deletedadenovirus expression system in order to explore their in vitro and invivo biological properties and anti tumor activity followingintratumoral injection. Of these, several fusion cytokines have showninteresting properties, which include the maintenance of biologicalactivity of the two cytokines engaged in the fusion and, importantly, areduced toxicity (e.g. mIL2/proIL18 and mIL7/IL2). Moreover, the presentinvention illustrates that a number of the described Ad-fusion cytokinesare effective for treating tumors of various origins (e.g. Ad-mIL-7/IL-2, Ad-mIL-21/mL-2, Ad-mIFNg/mL-2 and Ad-mIL-2/mproIL-18).

More particularly, the results illustrated in the present examples showthat the location of each cytokine with respect to each other mayinfluence expression and biological activity at least in adenoviralsystem. In this consideration, secretion and activity of the IL-2 andIL-18 entities was found much more efficient when IL-2 is located at theN-terminus of the fusion, (IL-2/IL-18 fusion) than when IL-2 is placedat the C-terminus of IL-18. In contrast, more efficient fusion cytokinesbetween IL-2 and IL-7 were obtained when IL-2 is located at theC-terminus of the IL-7 entity (see Examples 1 and 2).

Moreover, it is known that IL-18 is produced in a precursor form(proIL-18) initially. The IL-18 precursor should be cleaved by theenzyme Caspase-3/ICE to be secreted (Dinarello et al., 1999,Interleukin-18 Methods 19, 121-132). Examples 1 and 2 illustrate thatfusion cytokines incorporating pro-IL-18 are more effectively expressedthan those containing the mature IL-18. On this basis, one may assumethat upon expression of the IL-2/proIL-18 fusion cytokine, pro-IL18 iscorrectly folded and secreted, presumably as a result of theIL-2-associated signal sequence.

A mutation of IL-18 (K89A) was recently reported to augment thebiological activity of IL-18. IL18(K89A)-containing fusion cytokinesalso exhibit an improvement of functionality in all biological assaystested. More importantly, Example 3 shows that the IL18(K89A)-containingfusion cytokines display in addition a markedly reduced cytokine relatedtoxicity as assessed by Annexin V, Fas induction (AICD) or Vascular leak(VLS). Interestingly, biological activity of the mIL2/mpro-IL18(K89A)fusion cytokine appears to be maintained at a much lower and thus lessor non toxic protein concentration than required for biological activityof the individual cytokines. The reduced toxicity pattern obtained withthe mIL-2/proIL-18(K89A) fusion may be an effect of the structuralmodification of the cytokines engaged in the fusion protein. It is alsopossible that the murine fusion cytokine activates a specific populationof IL-2 receptor expressing effector cells, thus reducing the apparenttoxicity of recombinant IL-2 (Bensinger et al., 2004, J. Immunol. 172,5287-5296).

It is well known that T cell stimulation of individual IL-2 or IL-18cytokine to produce IFNg requires the pre-activation of splenic T cellsby Con-A (Osaki et al., 1998, J. Immunol. 160, 1742-1749; Osaki et al.,1999, Gene Ther. 6, 808-815; Hwang et al., 2004, Cancer Gene Ther. 11,397.407) Interestingly, as shown in example 2, the mIL-2/mproIL-18fusion cytokine does not require pre-stimulation of T cells for thisactivity. Thus, not only are IL-2 and IL-18 biological activitiesmaintained and cytokine related toxicity reduced, but the mIL-2/proIL-18fusion protein appears to have a novel activity which either individualcytokine is unable to exert.

As illustrated in Example 4, a very effective anti-tumor protection hasbeen obtained following intratumoral injection ofAd-mIL-2/mproIL-18(K89A) in all tumor models tested, including the veryaggressive B16F10 model. Antitumor activity provided byAd-mIL-2/mproIL-18(K89A) was much higher than that obtained followingintratumoral injection of either Ad-IL-2 or Ad-proIL-18 alone or thecombination of the two individual constructs. Depletion experimentsdemonstrate clearly that both the innate (NK, cells) immune system aswell as the adaptive (CD8) immune response are involved in thistherapeutic effect. Immuno-histological analysis of the injected tumorsindicate that the anti-tumor activity provided by Ad-mIL2/pro-IL18(K89A)is associated with infiltration of activated T cells and antigenpresenting cells. Surprisingly, tumors injected withAd(mIL-2/proIL-18(K89A) are highly positive for von Willebrand factor,suggesting increased vascularization. While vascularization of tumors isnormally associated with poor prognosis, in this case it may beassociated with an increased infiltration by immune effector cells.Moreover, mIL-2/proIL-18(K89A) fusion cytokine displays a reduced AICDactivity which seems to be crucial in the induction of tumor specific Tcells (Saff et al., 2004, J. Immunol. 172, 6598-6606).

In conclusion, on the basis of the above-discussed results, the fusioncytokines of the present invention have a great potential for bothincreasing the therapeutic activity of cytokines, and reducing the toxicside effects.

1-41. (canceled)
 42. An isolated infectious viral particle comprising apolynucleotide sequence encoding a fusion protein, wherein said fusionprotein comprises the amino acid sequence of SEQ ID NO:
 1. 43. Theisolated infectious viral particle of claim 42, wherein: said isolatedinfectious viral particle comprises a viral vector; said viral vectorcomprises a polynucleotide sequence encoding a fusion protein; and saidfusion protein comprises the amino acid sequence of SEQ ID NO:
 1. 44.The infectious viral particle of claim 43, wherein said viral vector isselected from the group consisting of baculoviral vectors, papoviralvectors, herpes viral vectors, adenoviral vectors, adenovirus-associatedviral vectors, poxyiral vectors, foamy viral vectors, and retroviralvectors.
 45. The isolated infectious viral particle of claim 44, whereinsaid viral vector is an adenoviral vector.
 46. The isolated infectiousviral particle of claim 45, wherein: said adenoviral vector is an E1-and E3-deleted replication-defective adenoviral vector comprising apolynucleotide sequence encoding a fusion protein inserted inreplacement of the E1 region; said fusion protein comprises the aminoacid sequence of SEQ ID NO: 1; and said polynucleotide sequence encodinga fusion protein is placed under the control of the CMV promoter. 47.The isolated infectious viral particle of claim 43, wherein said viralvector further comprises one or more transgenes encoding (i) a tumorproliferation inhibitor and/or (ii) at least one antigen.
 48. Theisolated infectious viral particle of claim 47, wherein said tumorproliferation inhibitor is a fusion protein encoding a two domain enzymepossessing both CDase and UPRTase activities.
 49. The isolatedinfectious viral particle of claim 47, wherein said antigen is a HPVantigen selected from the group consisting of E5, E6, E7, L1, L2, andcombinations thereof.
 50. The isolated infectious viral particle ofclaim 49, wherein said HPV antigen is a membrane-anchored form of anon-oncogenic variant of the early HPV-16 E6 and/or E7 antigen.
 51. Anisolated host cell comprising the infectious viral particle of claim 42.52. An isolated host cell comprising the infectious viral particle ofclaim
 43. 53. A composition comprising the infectious viral particle ofclaim 42 and a pharmaceutically acceptable vehicle.
 54. A compositioncomprising the infectious viral particle of claim 43 and apharmaceutically acceptable vehicle.
 55. A composition comprising theisolated host cell of claim 51 and a pharmaceutically acceptablevehicle.
 56. A composition comprising the isolated host cell of claim 52and a pharmaceutically acceptable vehicle.
 57. A process for producingthe infectious viral particle of claim 42, comprising the steps of: (a)introducing a viral vector into a suitable cell line, wherein said viralvector comprises a polynucleotide sequence encoding a fusion protein,and said fusion protein comprises the amino acid sequence of SEQ ID NO:1; (b) culturing said cell line under suitable conditions so as to allowthe production of said infectious viral particle; (c) recovering theproduced infectious viral particle from the culture of said cell line;and (d) optionally purifying said recovered infectious viral particle.